2,4-Pyrimidinediamine Compounds and Prodrugs and Their Uses

ABSTRACT

The present disclosure provides biologically active 2,4-pyrimidinediamine compounds of formulae (I)-(III): 
     
       
         
         
             
             
         
       
     
     and salts thereof, compositions comprising these compounds, and methods of using these compounds in a variety of applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/950,612, which claims the priority of U.S. ProvisionalPatent Application No. 61/263,169, each of which is hereby incorporatedherein by reference in its entirety.

BACKGROUND

1. Field of the disclosure

The present disclosure relates to biologically active2,4-pyrimidinediamine compounds and prodrugs thereof, pharmaceuticalcompositions comprising these compounds, intermediates and syntheticmethods of making these compounds and methods of using these compoundsand compositions in a variety of contexts, such as in the treatment orprevention of various diseases.

2. Description of the Related Art

Crosslinking of Fc receptors, such as the high affinity receptor for IgE(FccRI) and/or the high affinity receptor for IgG (FcγRI) activates asignaling cascade in mast, basophil and other immune cells that resultsin the release of chemical mediators responsible for numerous adverseevents. For example, such crosslinking leads to the release of preformedmediators of Type I (immediate) anaphylactic hypersensitivity reactions,such as histamine, from storage sites in granules via degranulation. Italso leads to the synthesis and release of other mediators, includingleukotrienes, prostaglandins and platelet-activating factors (PAFs),that play important roles in inflammatory reactions. Additionalmediators that are synthesized and released upon crosslinking Fcreceptors include cytokines and nitric oxide.

The signaling cascade(s) activated by crosslinking Fc receptors such asFcεRI and/or FcγRI includes an array of cellular proteins. Among themost important intracellular signal propagators are the tyrosinekinases. One important tyrosine kinase involved in the signaltransduction pathways associated with crosslinking the FcεRI and/orFcγRI receptors, as well as other signal transduction cascades, is Sykkinase (see Valent et al., 2002, Intl. J. Hematol. 75(4):257-362 forreview).

The mediators released as a result of FcεRI and FcγRI receptorcross-linking are responsible for, or play important roles in, themanifestation of numerous adverse events. Recently, various classes of2,4-pyrimidinediamine compounds have been discovered that inhibit theFcεRI and/or FcγRI signaling cascades, and that have myriad therapeuticuses. See, e.g., U.S. patent application Ser. No. 10/355,543 filed Jan.31, 2003 (US 2004/0029902A1), international patent application SerialNo. PCT/US03/03022 filed Jan. 31, 2003 (WO 03/063794), U.S. patentapplication Ser. No. 10/631,029 filed Jul. 29, 2003 (US 2007/0060603),international patent application no. PCT/US03/24087 (WO 2004/014382),U.S. patent application Ser. No. 10/903,263 filed Jul. 30, 2004(US2005/0234049), and international patent application no.PCT/US2004/24716 (WO 2005/016893), each of which is hereby incorporatedherein by reference in its entirety. While many of these compoundsexhibit good bioavailability properties, in some instances it may bedesirable to tailor their solubility or other properties such that theirbioavailability via specified routes of administration is optimized.

International patent application no. PCT/US03/03022 filed Jan. 31, 2003(WO 03/063794), international patent application no. PCT/US07/85313filed Nov. 20, 2007 (WO 2008/064274), and international patentapplication no. PCT/US06/01945 filed Jan. 19, 2006 (WO 2006/078846),each of which is hereby incorporated herein by reference in itsentirety, disclose a class of 2,4-pyrimidinediamine compounds andprodrugs thereof as being useful in a variety of in vitro and in vivocontexts, including in the treatment and/or prevention of diseasesmediated, at least in part, by the activation of Fc receptor signalingcascades. While these compounds are useful in a variety of in vitro andin vivo contexts, there remains a need for compounds with improvedeffects and increased duration of actions.

SUMMARY OF THE DISCLOSURE

In a broad aspect, the disclosure provides 2,4-pyrimidinediaminecompounds and prodrugs thereof that have myriad biological activities,and hence therapeutic uses, compositions comprising the compounds andprodrugs, methods and intermediates useful for synthesizing thecompounds and prodrugs and methods of using the compounds and prodrugsin a variety of in vitro and in vivo contexts, including in thetreatment and/or prevention of diseases mediated, at least in part, bythe activation of Fc receptor signaling cascades.

Thus, one aspect of the disclosure provides a compound of formula (I):

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides a compound of formula (II):

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides a compound of formula (III):

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides pharmaceutical compositionscomprising the compounds or salts of the disclosure and an appropriatecarrier, excipient or diluent. The exact nature of the carrier,excipient or diluent will depend upon the desired use for thecomposition, and may range from being suitable or acceptable forveterinary uses to being suitable or acceptable for human use. Thecomposition may optionally include one or more additional compounds.

Another aspect of the disclosure provides a method of inhibiting celldegranulation in a subject, comprising administering to the subject apharmaceutically effective amount of a compound, salt or composition ofthe disclosure effective to inhibit degranulation.

Yet another aspect of the disclosure provides a method for treating orpreventing a disease selected from an allergic disease, low gradescarring, a disease associated with tissue destruction, a diseaseassociated with tissue inflammation, inflammation and scarring,comprising administering to the subject a pharmaceutically effectiveamount of a compound, salt or composition of the disclosure.

In one aspect, the disclosure provides a method of treating rheumatoidarthritis in a subject, comprising administering to a subject sufferingfrom rheumatoid arthritis a pharmaceutically effective amount of acompound, salt or composition of the disclosure.

Another aspect of the disclosure provides a method of inhibiting anactivity of a Syk kinase in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound, salt orcomposition of the disclosure effective to inhibit the Syk kinaseactivity.

In another aspect, the disclosure provides a method of inhibiting an Fcreceptor signal transduction cascade in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound, salt or composition of the disclosure effective to inhibit theFc receptor signal transduction cascade. Fc receptor is selected fromFcαRI, FcγRI, FcγRIII and FcεRI.

Another aspect of the disclosure provides a method of treating orpreventing an autoimmune disease in a subject, and/or one or moresymptoms associated therewith, comprising administering to the subject apharmaceutically effective amount of a compound, salt or composition ofthe disclosure effective to treat or prevent the autoimmune disease.

Another aspect of the disclosure provides a method of treating a cellproliferative disorder in a subject, comprising administering to asubject suffering from a cell proliferative disorder a pharmaceuticallyeffective amount of a compound, salt or composition according to thedisclosure.

Another aspect of the disclosure provides a method of regulating orinhibiting Syk kinase in a cell comprising contacting a Syk kinase or acell comprising a Syk kinase with a compound or salt of the disclosure.

Another aspect of the disclosure provides a method of regulating orinhibiting the Fc receptor signaling cascade comprising contacting acell expressing an Fc receptor with a compound or salt of thedisclosure.

Another aspect of the disclosure provides a method of regulating orinhibiting degranulation of a cell comprising contacting a cell thatdegranulates with a compound or salt of the disclosure.

Another aspect of the disclosure provides a method of regulating orinhibiting the signal transduction cascade comprising contacting aSyk-dependent receptor or a cell expressing a Syk-dependent receptorwith a compound or salt of the disclosure.

DETAILED DESCRIPTION

In one embodiment, the disclosure providesN4-(2,2-dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-[6-morpholinopyridin-3-yl]-2,4-pyrimidinediamine,and pharmaceutically acceptable salts thereof. This compound has formula(I):

In another embodiment, the disclosure providesN4-[2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl]-5-fluoro-N2-(6-morpholinopyridin-3-yl)-2,4-pyrimidinediamine,and pharmaceutically acceptable salts thereof. This compound has formula(II):

In yet another embodiment, the disclosure providesN4-[2,2-Dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl]-5-fluoro-N2-(4-morpholinophenyl)-2,4-pyrimidinediamine,and pharmaceutically acceptable salts thereof. This compound has formula(III):

In the compounds of formulae (II) and (III), the methyl dihydrogenphosphate substituted at the 4-position of the pyrido[3,2-b]oxazine canact as a progroup that metabolizes or otherwise transforms underconditions of use to yield the active 2,4-pyrimidinediamine compounds.The phosphate moieties can be cleaved in vitro by enzymes such asesterases, lipases and/or phosphatases. Such enzymes are prevalentthroughout the body, residing in, for example, the stomach and digestivetract, blood and/or serum, and in virtually all tissues and organs. Suchphosphate-containing progroups will generally increase thewater-solubility of the underlying active 2,4-pyrimidinediaminecompound, making such phosphate-containing compounds suitable for modesof administration where water-solubility is desirable, such as, forexample, oral, buccal, intravenous, intramuscular and ocular modes ofadministration.

In one embodiment of the disclosure, incorporation of a heavy atom,particularly substitution of hydrogen with deuterium, into the compoundsas described above with respect to any of formulae (I)-(III) can giverise to an isotope effect that can alter the pharmacokinetics of thecompound. Stable isotope labeling of a compound of the disclosure canalter its physicochemical properties such as pKa and lipid solubility.These changes may influence the fate of the compound at different stepsalong its passage through the body. Absorption, distribution, metabolismor excretion can be changed. The deuterated compound can have anincreased effect and an increased duration of action on mammals at lowerconcentration than the undeuterated compound.

Deuterium has a natural abundance of about 0.015%. Accordingly, forapproximately every 6,500 hydrogen atoms occurring in nature, there isone deuterium atom. Disclosed herein are compounds enriched in deuteriumat one or more positions. Thus, deuterium containing compounds of thedisclosure have deuterium at one or more positions (as the case may be)in an abundance of greater than 0.015%.

In one embodiment, a compound as described above with respect to any offormulae (I)-(XII), at a position designated as having deuterium, has aminimum isotopic enrichment factor of at least 2000 (30% deuteriumincorporation) at each atom designated as deuterium in the compound, orat least 3000 (45% deuterium incorporation).

In other embodiments, a compound as described above with respect to anyof formulae (I)-(III) has an isotopic enrichment factor for eachdesignated deuterium atom of at least 3500 (52.5% deuteriumincorporation at each designated deuterium atom), at least 4000 (60%deuterium incorporation), at least 4500 (67.5% deuterium incorporation),at least 5000 (75% deuterium incorporation), at least 5500 (82.5%deuterium incorporation), at least 6000 (90% deuterium incorporation),at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97%deuterium incorporation), at least 6600 (99% deuterium incorporation),or at least 6633.3 (99.5% deuterium incorporation).

In another embodiment, the disclosure provides pharmaceuticallyacceptable salts of compounds as described above with respect to any offormulae (I)-(III). Generally, pharmaceutically acceptable salts arethose salts that retain substantially one or more of the desiredpharmacological activities of the parent compound and which are suitablefor administration to humans. Pharmaceutically acceptable salts includeacid addition salts formed with inorganic acids or organic acids.Inorganic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation,hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydriodic,etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.Organic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation, aceticacid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid,propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolicacid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamicacid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like. Forexample, in one embodiment, the salt is or di-trifluoroacetic acid salt,methanesulfonic acid salt, p-toluenesulfonic acid salt, hydrochloridesalt, benzenesulfonic acid salt, or an ethanesulfonic acid salt.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by aninorganic ion (e.g., an alkali metal ion such as Na⁺, K⁺ or Li⁺, analkaline earth ion such as Ca²⁺ or Mg²⁺, an aluminum ion, or an ammoniumion) or coordinates with an organic base (e.g., ethanolamine,diethanolamine, triethanolamine, lysine, choline, N-methylglucamine,morpholine, piperidine, dimethylamine, diethylamine, or the like).

Specific exemplary salts include, but are not limited to, mono- anddi-sodium salts, mono- and di-potassium salts, mono- and di-lithiumsalts, mono- and di-alkylamino salts, mono- and di-ammonium salts,mono-magnesium salts, mono-calcium salts. Such salts can be especiallyuseful when the compound includes a free phosphate (i.e., —P(O)(OH)₂).

The compounds described herein, as well as the salts thereof, may alsobe in the form of hydrates, solvates and N-oxides, as are well-known inthe art.

Many of the compounds described herein, and in particular compounds asdescribed above with respect to any of formulae (I)-(III), are potentinhibitors of degranulation of immune cells, such as mast, basophil,neutrophil and/or eosinophil cells or metabolize to yield2,4-pyrimidinediamine compounds that potent inhibitors of degranulationof immune cells, such as mast, basophil, neutrophil and/or eosinophilcells. Thus, in still another aspect, the present disclosure providesmethods of regulating, and in particular inhibiting, degranulation ofsuch cells. The method generally involves contacting a cell thatdegranulates with an amount of a suitable compound described herein, oran acceptable salt thereof, effective to regulate or inhibitdegranulation of the cell. The method may be practiced in in vitrocontexts or in in vivo contexts as a therapeutic approach towards thetreatment or prevention of diseases characterized by, caused by orassociated with cellular degranulation.

While not intending to be bound by any theory of operation, biochemicaldata confirm that many 2,4-pyrimidinediamine compounds exert theirdegranulation inhibitory effect, at least in part, by blocking orinhibiting the signal transduction cascade(s) initiated by crosslinkingof the high affinity Fc receptors for IgE (“FcεRI”) and/or IgG (“FcγRI”)(see, e.g., U.S. patent application Ser. No. 10/631,029 filed Jul. 29,2003 (US 2007/0060603), international patent application no.PCT/US03/24087 (WO2004/014382), U.S. patent application Ser. No.10/903,263 filed Jul. 30, 2004 (US2005/0234049), and internationalpatent application no. PCT/US2004/24716 (WO 2005/016893), thedisclosures of which are hereby incorporated herein by reference intheir entireties). Indeed, these active 2,4-pyrimidinediamine compoundsare potent inhibitors of both FcεRI-mediated and FcγRI-mediateddegranulation. As a consequence, the compounds described herein may beused to inhibit these Fc receptor signaling cascades in any cell typeexpressing such FcεRI and/or FcγRI receptors including but not limitedto macrophages, mast, basophil, neutrophil and/or eosinophil cells.

The methods also permit the regulation of, and in particular theinhibition of, downstream processes that result as a consequence ofactivating such Fc receptor signaling cascade(s). Such downstreamprocesses include, but are not limited to, FcεRI-mediated and/orFcγRI-mediated degranulation, cytokine production and/or the productionand/or release of lipid mediators such as leukotrienes andprostaglandins. The method generally involves contacting a cellexpressing an Fc receptor, such as one of the cell types discussedabove, with an amount of a compound described herein, or an acceptablesalt thereof, effective to regulate or inhibit the Fc receptor signalingcascade and/or a downstream process effected by the activation of thissignaling cascade. The method may be practiced in in vitro contexts orin in vivo contexts as a therapeutic approach towards the treatment orprevention of diseases characterized by, caused by or associated withthe Fc receptor signaling cascade, such as diseases effected by therelease of granule specific chemical mediators upon degranulation, therelease and/or synthesis of cytokines and/or the release and/orsynthesis of lipid mediators such as leukotrienes and prostaglandins.

In yet another aspect, the present disclosure provides methods oftreating and/or preventing diseases characterized by, caused by orassociated with the release of chemical mediators as a consequence ofactivating Fc receptor signaling cascades, such as FcεRI and/orFcγRI-signaling cascades. The methods may be practiced in animals inveterinary contexts or in humans. The methods generally involveadministering to an animal subject or a human an amount of a compound asdescribed above with respect to any of formulae (I)-(III), or anacceptable salt thereof, effective to treat or prevent the disease. Asdiscussed previously, activation of the FcεRI or FcγRI receptorsignaling cascade in certain immune cells leads to the release and/orsynthesis of a variety of chemical substances that are pharmacologicalmediators of a wide variety of diseases. Any of these diseases may betreated or prevented according to the methods described herein.

Many of the compounds of the disclosure are also potent inhibitors ofthe tyrosine kinase Syk kinase. Thus, in still another aspect, thepresent disclosure provides methods of regulating, and in particularinhibiting, Syk kinase activity. The method generally involvescontacting a Syk kinase or a cell comprising a Syk kinase with an amountof a suitable compound as described above with respect to any offormulae (I)-(III), or an acceptable salt thereof, effective to regulateor inhibit Syk kinase activity. In one embodiment, the Syk kinase is anisolated or recombinant Syk kinase. In another embodiment, the Sykkinase is an endogenous or recombinant Syk kinase expressed by a cell,for example a mast cell or a basophil cell. The method may be practicedin in vitro contexts or in in vivo contexts as a therapeutic approachtowards the treatment or prevention of diseases characterized by, causedby or associated with Syk kinase activity.

While not intending to be bound by any particular theory of operation,it is believed that such active 2,4-pyrimdinediamine compounds inhibitcellular degranulation and/or the release of other chemical mediatorsprimarily by inhibiting Syk kinase that gets activated through the gammachain homodimer of FcεRI. This gamma chain homodimer is shared by otherFc receptors, including FcγRI, FcγRIII and FcαRI. For all of thesereceptors, intracellular signal transduction is mediated by the commongamma chain homodimer. Binding and aggregation of those receptorsresults in the recruitment and activation of tyrosine kinases such asSyk kinase. As a consequence of these common signaling activities,compounds as described above with respect to any of formulae (I)-(III)may be used to regulate, and in particular inhibit, the signalingcascades of Fc receptors having this gamma chain homodimer, such asFcεRI, FcγRI, FcγRIII and FcαRI, as well as the cellular responseselicited through these receptors.

Syk kinase is known to play a critical role in other signaling cascades.For example, Syk kinase is an effector of B-cell receptor (BCR)signaling and is an essential component of integrin beta(1), beta(2) andbeta(3) signaling in neutrophils. Active 2,4-pyrimidinediamine compoundsthat are potent inhibitors of Syk kinase can be used to regulate, and inparticular inhibit, any signaling cascade where Syk plays a role, suchas, fore example, the Fc receptor, BCR and integrin signaling cascades,as well as the cellular responses elicited through these signalingcascades. Thus, compounds as described above with respect to any offormulae (I)-(III) can be used to regulate such activities. Theparticular cellular response regulated or inhibited will depend, inpart, on the specific cell type and receptor signaling cascade, as iswell known in the art. Non-limiting examples of cellular responses thatmay be regulated or inhibited with such compounds include a respiratoryburst, cellular adhesion, cellular degranulation, cell spreading, cellmigration, phagocytosis (e.g., in macrophages), calcium ion flux (e.g.,in mast, basophil, neutrophil, eosinophil and B-cells), plateletaggregation, and cell maturation (e.g., in B-cells).

Thus, in another aspect, the present disclosure provides methods ofregulating, and in particular inhibiting, signal transduction cascadesin which Syk plays a role. The method generally involves contacting aSyk-dependent receptor or a cell expressing a Syk-dependent receptorwith an amount of a suitable compound described herein, or an acceptablesalt thereof, effective to regulate or inhibit the signal transductioncascade. The methods may also be used to regulate, and in particularinhibit, downstream processes or cellular responses elicited byactivation of the particular Syk-dependent signal transduction cascade.The methods may be practiced to regulate any signal transduction cascadewhere Syk is now known or later discovered to play a role. The methodsmay be practiced in in vitro contexts or in in vivo contexts as atherapeutic approach towards the treatment or prevention of diseasescharacterized by, caused by or associated with activation of theSyk-dependent signal transduction cascade. Non-limited examples of suchdiseases include those previously discussed.

Recent studies have shown that activation of platelets by collagen ismediated through the same pathway used by immune receptors, with animmunoreceptor tyronsine kinase motif on the FcRy playing a pivotalrole, and also that FcRy plays a pivotal role in the generation ofneointimal hyperplasia following balloon injury in mice, most likelythrough collagen-induced activation of platelets and leukocyterecruitment. Thus, the compounds described herein can also be used toinhibit collagen-induced platelet activation and to treat or preventdiseases associated with or caused by such platelet activation, such as,for example, intimal hyperplasia and restenosis following vascularinjury.

Therapeutic Applications

Compounds as described above with respect to any of formulae (I)-(III)are useful for treating a disease or disorder that is mediated through,or exacerbated by, the activity of a Syk in a subject in need oftreatment. The present disclosure provides methods of treatingconditions such as inflammatory conditions or diseases, autoimmunediseases, cell proliferative disorders, and degenerative bone disordersin a subject by administering an effective amount of a compound asdescribed above with respect to any of formulae (I)-(III), including asalt or solvate thereof.

Inflammatory Conditions

Accordingly, the present disclosure provides methods of treating aninflammatory condition or disease in a subject by administering aneffective amount of a subject compound, including a salt or solvatethereof. Inflammatory conditions contemplated for therapy include acuteand chronic inflammation mediated or exacerbated by Syk activity.

The subject compounds can be used to treat a variety of inflammatoryconditions or diseases in which an inflammatory response is associatedwith the condition or disease. Diagnosis and clinical indications ofsuch diseases and conditions will be well known to the skilled artisan,and guidance is provided in various reference works, such as The MerckManual of Diagnosis and Therapy, 1999, 17^(th) Ed., John Wiley & Sons;and International Classification of Disease and Related Health Problems(ICD 10), 2003, World Health Organization.

The disclosure provides methods of regulating or inhibiting signaltransduction cascades in which Syk plays a role. The method generallyinvolves contacting a Syk-dependent receptor or a cell expressing aSyk-dependent receptor with an amount of a subject compound effective toregulate or inhibit the signal transduction cascade. The methods mayalso be used to regulate or inhibit downstream processes or cellularresponses elicited by activation of the particular Syk-dependent signaltransduction cascade. The methods may be practiced in in vitro contextsor in in vivo contexts as a therapeutic approach towards the treatmentor prevention of diseases characterized by, caused by or associated withactivation of the Syk-dependent signal transduction cascade.

Syk is involved in release of preformed mediators of atopic and/or TypeI hypersensitivity reactions (e.g., histamine, proteases such astryptase, etc.) via the degranulation process in mast cells and basophilcells. Such atopic or Type I hypersensitivity reactions include, but arenot limited to, anaphylactic reactions to environmental and otherallergens (e.g., pollens, insect and/or animal venoms, foods, drugs,contrast dyes, etc.), anaphylactoid reactions, hay fever, allergicconjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis,eczema, urticaria, mucosal disorders, tissue disorders, and certaingastrointestinal disorders.

The immediate release of the preformed mediators via degranulation isfollowed by the release and/or synthesis of a variety of other chemicalmediators, including, but not limited to, platelet activating factor(PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novosynthesis and release of cytokines such as TNFα, IL-4, IL-5, IL-6,IL-13, etc. These “late stage” mediators can be responsible for thechronic symptoms of the above-listed atopic and Type I hypersensitivityreactions, and in addition are chemical mediators of inflammation andinflammatory conditions, including, but not limited to, osteoarthritis,inflammatory bowel disease, ulcerative colitis, Crohn's disease,idiopathic inflammatory bowel disease, irritable bowel syndrome, spasticcolon, low grade scarring, scleroderma, increased fibrosis, keloids,post-surgical scars, pulmonary fibrosis, vascular spasms, migraine,reperfusion injury, post myocardial infarction, and sicca complex orsyndrome. All of these diseases may be treated or prevented according tothe methods described herein.

Additional diseases which can be treated or prevented according to thesubject methods include diseases associated with basophil cell and/ormast cell pathology. Examples of such diseases include, but are notlimited to, diseases of the skin such as scleroderma, cardiac diseasessuch as post myocardial infarction, pulmonary diseases such as pulmonarymuscle changes or remodeling and chronic obstructive pulmonary disease(COPD, and diseases of the gut such as inflammatory bowel syndrome(spastic colon).

Certain inflammatory diseases or disorders that can be treated using thesubject compound include, but not limited to, asthma, COPD, lunginflammation, chronic granulomatous diseases such as tuberculosis,leprosy, sarcoidosis, and silicosis, nephritis, amyloidosis, rheumatoidarthritis, ankylosing spondylitis, chronic bronchitis, scleroderma,lupus, polymyositis, appendicitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, psoriasis, pelvic inflammatory disease,irritable bowel syndrome, orbital inflammatory disease, thromboticdisease, and inappropriate allergic responses to environmental stimulisuch as poison ivy, pollen, insect stings and certain foods, includingatopic dermatitis and contact dermatitis.

Because the exemplary compounds inhibit the FcεRI and/or FcyR signalcascades that lead to degranulation of immune cells such as mast cells,such compounds can be used to inhibit the development and progression ofatherosclerosis and associated symptoms. For example, activation of theIgE receptor signal transduction pathway leads to degranulation of thecells and consequent release and/or synthesis of a host of chemicalmediators, including histamine, proteases (e.g., tryptase and chymase),lipid mediators such as leukotrienes (e.g., LTC4), platelet-activatingfactor (PAP) and prostaglandins (e.g., PGD2) and a series of cytokines,including TNF-a, IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β.The release and/or synthesis of these mediators from mast cells can leadto degradation of the extracellular matrix, deposition of fatty streaksin the vasculature and rupture of existing atherosclerotic plaques.Accordingly, inhibition of mast cell degranulation using the presentlydisclosed compounds can be used to treat atherosclerosis.

The subject compounds can be used, either independently or incombination with other anti-inflammatory compositions, as discussedbelow.

Autoimmune Diseases

The present disclosure provides methods of treating an autoimmunedisease in a subject by administering an effective amount of a subjectcompound, including a salt or solvate thereof.

The subject compounds can also be used to treat or prevent autoimmunediseases and/or symptoms of such diseases. Autoimmune diseases that canbe treated or prevented with the subject compounds include thosediseases that are commonly associated with nonanaphylactichypersensitivity reactions (Type II, Type III and/or Type IVhypersensitivity reactions) and/or those diseases that are mediated, atleast in part, by activation of the FcγR signaling cascade in monocytecells. Such autoimmune disease include autoimmune diseases that arefrequently designated as single organ or single cell-type autoimmunedisorders and autoimmune disease that are frequently designated asinvolving systemic autoimmune disorder. Non-limiting examples ofdiseases frequently designated as single organ or single cell-typeautoimmune disorders include: Hashimoto's thyroiditis, autoimmunehemolytic anemia, autoimmune atrophic gastritis of pernicious anemia,autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture'sdisease, autoimmune thrombocytopenia, sympathetic ophthalmia, myastheniagravis, Graves' disease, primary biliary cirrhosis, chronic aggressivehepatitis, ulcerative colitis and membranous glomerulopathy.Non-limiting examples of diseases often designated as involving systemicautoimmune disorder include: systemic lupus erythematosis, rheumatoidarthritis, Sjogren's syndrome, Reiter's syndrome,polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa,multiple sclerosis and bullous pemphigoid.

As a certain example of a treatment, rheumatoid arthritis is thought tobe an autoimmune disease that commonly affects the joints in apolyarticular manner (polyarthritis). The disease is characterized bychronically inflamed synovium that is densely crowded with lymphocytes.Chronic inflammatory condition arising from an autoimmune reaction canlead to led to erosion and destruction of the joint surface, whichimpairs the range of joint movement and leads to deformity. The subjectcompounds can be used to treat or ameliorate any one, several or all ofthese symptoms of rheumatoid arthritis.

The subject compounds can be used, either independently or incombination with other anti-inflammatory compositions, as discussedbelow.

Cellular Proliferation Disorders

Although the art suggests that Syk may act as a tumor suppressor, thepresent disclosure is based, in part, on indications that Syk functionscontrary to that posited role. For instance, forced expression of Sykkinase in tumor cells does not appear to reverse the transformedphenotype of tumor cells. To the contrary, it is suggested herein thatSyk acts in an oncogenic capacity to promote and/or maintain cellproliferation. With this perspective on the role of Syk, the presentdisclosure provides methods of treating a cell proliferative disorder ina subject by administering an effective amount of a subject compound,including a salt or solvate thereof.

Generally, cell proliferative disorders treatable with the subjectcompound disclosed herein relate to any disorder characterized byaberrant cell proliferation. These include various tumors and cancers,benign or malignant, metastatic or non-metastatic. Specific propertiesof cancers, such as tissue invasiveness or metastasis, can be targetedusing the methods described herein. Cell proliferative disorders includea variety of cancers, including, but not limited to, breast cancer,ovarian cancer, renal cancer, gastrointestinal cancer, kidney cancer,bladder cancer, pancreatic cancer, lung squamous carcinoma, andadenocarcinoma.

In certain instances, the cell proliferative disorder treated is ahematopoietic neoplasm, which is aberrant growth of cells of thehematopoietic system.

In certain instances, the hematopoietic neoplasm is a lymphoid neoplasm,where the abnormal cells are derived from and/or display thecharacteristic phenotype of cells of the lymphoid lineage. Lymphoidneoplasms can be subdivided into B-cell neoplasms, T and NK-cellneoplasms, and Hodgkin's lymphoma. B-cell neoplasms can be furthersubdivided into precursor B-cell neoplasm and mature/peripheral B-cellneoplasm. Certain B-cell neoplasms are precursor B-lymphoblasticleukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) whilecertain mature/peripheral B-cell neoplasms are B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-celllymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma,extranodal marginal zone B-cell lymphoma of MALT type, nodal marginalzone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuselarge B-cell lymphoma, mediastinal large B-cell lymphoma, primaryeffusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia. T-celland Nk-cell neoplasms can be further subdivided into precursor T-cellneoplasm and mature (peripheral) T-cell neoplasms. A certain precursorT-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia(precursor T-cell acute lymphoblastic leukemia) while certain mature(peripheral) T-cell neoplasms are T-cell prolymphocytic leukemia T-cellgranular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-celllymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type,enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosisfungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell,primary cutaneous type, peripheral T-cell lymphoma, not otherwisecharacterized, angioimmunoblastic T-cell lymphoma, anaplastic large-celllymphoma, T/null cell, primary systemic type. Another member of lymphoidneoplasms is Hodgkin's lymphoma, also referred to as Hodgkin's disease.Certain diagnoses of this class that include, among others, nodularlymphocyte-predominant Hodgkin's lymphoma, and various classical formsof Hodgkin's disease, certain members of which are nodular sclerosisHodgkin's lymphoma (grades 1 and 2), lymphocyte-rich classical Hodgkin'slymphoma, mixed cellularity Hodgkin's lymphoma, and lymphocyte depletionHodgkin's lymphoma.

In certain instances, the hematopoietic neoplasm is a myeloid neoplasm.This group includes a large class of cell proliferative disordersinvolving or displaying the characteristic phenotype of the cells of themyeloid lineage. Myeloid neoplasms can be subdivided intomyeloproliferative diseases, myelodysplastic/myeloproliferativediseases, myelodysplastic syndromes, and acute myeloid leukemias.Certain myeloproliferative diseases include chronic myelogenous leukemia(e.g., Philadelphia chromosome positive (t(9; 22)(qq34; q11)), chronicneutrophilic leukemia, chronic eosinophilic leukemialhypereosinophilicsyndrome, chronic idiopathic myelofibrosis, polycythemia vera, andessential thrombocythemia. Certain myelodysplastic/myeloproliferativediseases include chronic myelomonocytic leukemia, atypical chronicmyelogenous leukemia, and juvenile myelomonocytic leukemia. Certainmyelodysplastic syndromes include refractory anemia, with ringedsideroblasts and without ringed sideroblasts, refractory cytopenia(myelodysplastic syndrome) with multilineage dysplasia, refractoryanemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, andmyelodysplastic syndrome with t(9; 12)(q22; p12) (TEL-Syk fusion).

In certain instances, the composition can be used to treat acute myeloidleukemias (AML), which represent a large class of myeloid neoplasmshaving its own subdivision of disorders. These subdivisions include,among others, AMLs with recurrent cytogenetic translocations, AML withmultilineage dysplasia, and other AML not otherwise categorized. CertainAMLs with recurrent cytogenetic translocations include, among others,AML with t(8; 21)(q22; q22), AML1(CBF-alpha)/ETO, acute promyelocyticleukemia (AML with t(15; 17)(q22; q11-12) and variants, PML/RAR-alpha),AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(16;16)(p13; q11), CBFb/MYH11X), and AML with 11q23 (MLL) abnormalities.Certain AML with multilineage dysplasia are those that are associatedwith or without prior myelodysplastic syndrome. Other acute myeloidleukemias not classified within any definable group include, AMLminimally differentiated, AML without maturation, AML with maturation,acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroidleukemia, acute megakaryocytic leukemia, acute basophilic leukemia, andacute panmyelosis with myelofibrosis.

In certain instances, cell proliferative disorders include virallymediated tumors. These can arise from infection of cells by an oncogenicvirus that has a capability of transforming a normal cell into a tumorcell.

In certain instances, the virally mediated tumor can be associated withany virus that encodes an immunoreceptor tyrosine-based activation motif(ITAM) capable of modulating Syk activity. This motif can refer to aconserved amino acid sequence motif that functions by interacting withand activating nonreceptor tyrosine kinases. ITAM motifs are found in,among others, the p and y chains of FcεRI, the ε subunit of the T cellreceptor, and immunoglobulin β (Igβ) and Iga of the B cell receptor. Thecanonical sequence motif is typically Yxx(L/I)x₆₋₈Yxx(L/I), where xrepresents any amino acid.

Accordingly, in certain instances, the virally mediated tumor can beassociated with Kaposi's sarcoma (KS) associated herpes virus, alymphotropic virus implicated in Kaposi's sarcoma. The KS associatedherpes virus encodes a transmembrane protein termed KI having animmunoreceptor tyrosine-based activation motif (ITAM)-like sequence.

In certain instances, the virally mediated tumor can be associated withEpstein Barr Virus (EBV). Epstein Barr Virus is a member of theHerpesviridae family that, following primary infection, replicates inthe epithelial cells of the oropharynx and infect recirculating Blymphocytes. EBV infection can be associated with Burkitt's lymphoma,Hodgkin's lymphoma, and adult T cell leukemia.

In certain instances, the virally mediated tumor can be associated withHuman T-cell Lymphotropic Virus (HTLV-1 virus), a retrovirus in the sameclass of virus as HIV-1.

In certain instances, the virally mediated tumor can be associated withmammary tumor virus (MTV). ITAM sequences can be found within the Envgene of murine mammary tumor virus (MMTV), a B type retrovirusidentified as an etiological agent for breast cancer in mice. Murinemammary tumor virus-like sequences can be present in human cancers, suchas breast cancer and T cell lymphomas.

It is to be understood that use of subject composition for treatingvirally mediated tumors is not limited to tumors associated with theviruses specified above. As noted, any tumors associated with anoncogenic virus in which Syk is activated as part of its oncogenicmechanism, whether or not it involves ITAM sequences, can be targetedusing the subject compounds.

In certain instances, the subject compounds can be used for thetreatment of tumor metastasis. Metastasis is a characteristic ofmalignant tumor cells whereby tumor cells detach from its site of originand then spread to colonize at other sites. These secondary tumors canform in tissues unrelated to the cells from which the tumor cellsoriginate.

Various tumor types capable of metastasis can be treated with thesubject compounds. Such tumors include, but not limited to, breastcancer, ovarian cancer, renal cancer, gastrointestinal cancer, kidneycancer, bladder cancer, pancreatic cancer, lung squamous carcinoma, andadenocarcinoma. Therapeutic treatment to attenuate the metastasis ofestablished tumors can follow a diagnosis of metastasis. If no diagnosisof metastasis has been made, the subject compounds can be administeredprophylactically to reduce the probability of metastasis.

The subject compounds can be used, either independently or incombination with other chemotherapeutic compositions, as recognized inthe art.

Degenerative Bone Disorders

The present disclosure provides methods of treating a degenerative bonedisorder in a subject by administering an effective amount of a subjectcompound, including a salt or solvate thereof.

The subject compounds can be used for treating degenerative bonedisorders as well as prophylactic approaches for preventing bone lossthat can lead to increased fracture risk. These treatments are based onthe use of Syk inhibitors to attenuate or inhibit osteoclastogenesis andosteoclast activity, thereby decreasing or inhibiting the excessive boneloss associated with abnormal activity of osteoclasts. In addition, inthose degenerative bone disorders where inappropriate remodeling resultsin compromised bone integrity but without significant bone loss, anincrease in bone mass resulting from inhibition of bone resorption canincrease bone strength sufficiently to decrease the fracture risk. Thesubject compounds can be used independently or in combination with othermodulators of bone remodeling (i.e., antiresorptive agents andosteo-anabolic agents), for treatment as well as prophylaxis.

The diagnosis of a particular disorder can be based on clinicalpresentations typically used by those skilled in the art to diagnose thedisorder. As further discussed herein, other diagnostic criteria such asthe presence of biochemical and molecular markers of the disease, can beused independently or as a supplement to the examination of the clinicalpresentations. Standard diagnostic criteria can be found in variousreferences, including, by way of example and not limitation, the WorldHealth Organization's International Classification of Diseases, TenthRevision (lCD-i 0); Resnick, D., Diagnosis of Bone and Joint Disorders,4th Ed., W.B. Saunders Company (2002); and AACE Medical Guidelines forClinical Practice for the Prevention and Treatment of PostmenopausalOsteoporosis: 2001 Edition, with Selected Updates for 2003.

In certain instances, the subject compounds can be used to treat primaryosteoporosis, which is a loss of bone mass unrelated to any otherunderlying disease or illness. There are general types of primaryosteoporosis. Type I, also referred to as high turnover orpostmenopausal osteoporosis, is correlated with a decrease in hormonelevels secreted by the ovaries in the postmenopausal period. Type II,also referred to as low turnover or senile osteoporosis, can arise whenthe processes of bone resorption and bone formation are not coordinatedsuch that there is a net excess of bone resorption over bone formation.

Other forms of primary osteoporosis are idiopathic osteoporosis, anosteoporotic condition where there is no identifiable cause for the boneloss. Idiopathic osteoporosis can affect children and adults. Juvenileosteoporosis is osteoporosis occurring in children between the ages ofabout 8 and about 14 years of age.

In certain instances, the subject compounds can be used to treatosteodystrophy, a degeneration of bone resulting from compromised kidneyfunction. Clinical presentations of osteodystrophy can be in the form ofosteoporosis, osteomalacia, osteitis fibrosa, osteoscierosis,osteomalacia, and secondary hyperparathyroidism.

In certain instances, the subject compounds can be used to treat Paget'sDisease, also known as osteitis deformans.

In certain instances, the subject compounds can be used to treatperiodontal disease.

In certain instances, the subject compounds can be used to treatdegenerative bone disorders arising from a secondary condition, wherethe bone degeneration is a consequence of the underlying medicalcondition or disease. Thus, subject compounds can be administered tosubjects with the secondary condition to treat or prevent degenerativebone disorder associated with the secondary condition.

A certain secondary condition is encrinopathy, which is a conditioncharacterized by abnormal hormone secretion. Abnormal hormone secretioncan be either an increase or reduction in hormone levels. Varioushormones can affect bone metabolism, including but not limited to,estrogen, testosterone, growth hormone, calcitonin, parathyroid hormone,parathyroid hormone related protein, glucocorticoids, and calcitriol.Various forms of endocrinopathies are associated with loss of bone massand corresponding bone degeneration. In certain instances, the subjectcompounds can be used to treat bone degeneration arising fromhypercorticolism or an abnormal increase in the production ofglucocorticoids by the adrenal glands (e.g., Cushing's syndrome). Incertain instances, the subject compounds can be used to treat bonedegeneration arising from hypogonadism. In certain instances, the bonedegeneration treatable with the subject compounds can be bone lossassociated with destruction of one or both of the gonads, such as bysurgery (i.e., ovariectomy or oophorectomy). In certain instances, thesubject compounds can be used to treat bone degeneration arising fromhyperparathyroidism.

In certain instances, the methods can be directed to use of the subjectcompounds to treat bone degeneration associated with heritable geneticdisorders. Thus, subject compounds can be administered to subjects witha heritable genetic disorder to treat or prevent degenerative bonedisorder associated with the heritable genetic disorder. Inheritedgenetic disorders can arise from, among others, single gene inheritance,multifactorial or polygenic inheritance, chromosome abnormalities, andparental imprinting abnormalities. Various inherited geneticabnormalities affecting bone metabolism have been identified, including,osteogenesis imperfecta, homocystinurea, gonadal dysgenesis, andhypophosphatasia.

It is to be understood that the use of Syk inhibitors are not limited tothe degenerative bone disorders described herein, but may be applied todegenerative bone disorder characterized by a net excess of boneresorption over bone formation. This condition may arise from increasedosteoclastogenesis, increased osteoclast activation, decreasedosteoblastogeneis, decreased osteoblast activity, or a combination ofincreased osteclastogenesis and decreased osteoblastogenesis. Thus, themethods herein encompass treatments for degenerative bone disordersgenerally in which there is an imbalance of bone resorption over boneformation.

The subject compounds can be used, either independently or incombination with other bone modulating agents, as recognized in the art.In addition to the treatment of degenerative bone disorders, the subjectcompounds can be used, either independently or in combination with bonemodulating agents, as prophylaxis to prevent bone loss in subjects atrisk of bone loss and increased fracture risk.

Combination Therapy

The subject compounds may be administered individually or as compatiblecombinations along with an anti-inflammatory agent. Differentcombinations of the subject compounds may be used to adjustbioavailability, duration of effect, and efficacy for the particularinflammatory condition. Identifying appropriate combinations for thepurposes herein are within the skill of those in the art.

Steroidal Anti-inflammatory Agents

For treating inflammatory disorders, the subject compounds can beadministered in combination with an additional chemotherapeutic agent,such as an anti-inflammatory agent. In certain instances, theanti-inflammatory agent for use in combination with the presentlydisclosed compounds is a steroidal anti-inflammatory agent. As usedherein, “steroidal anti-inflammatory agent” or “anti-inflammatorysteroid” is a compound or composition based on a structure with asteroid nucleus and having anti-inflammatory activity, either alone orin combination with other agents. With the exception of vitamin Dcompounds, steroid compounds are derived from a steroid nucleus based ona saturated tetracyclic hydrocarbon,1,2-cyclopentanoperhydrophenanthrene, also referred to as sterane orgonane. Steroidal compounds include both naturally occurring andsynthetically produced steroidal compounds. Different groups of steroidcompounds include, among others, adrenocorticosteroids,estrogens/progestins, and androgens.

In certain instances, the steroidal anti-inflammatory agents areadrenocorticosteroids, which refer to steroidal compounds that arereleased from the adrenal cortex. These steroid compounds include thegroups of glucocorticosteroids and mineralocorticosteoids. As usedherein, adrenocorticosteroids also include various synthetic analogsthat display the biological properties displayed by the naturallyoccurring steroids. Certain structural features may enhanceanti-inflammatory activities of steroids, such as all trans steroidskeleton, presence of Δ⁴-3-keto, 11β-OH, 17β-OH, and substitutions at9α-, 6α-, 16α-positions, with F>Cl>Br>I.

In certain instances, the anti-inflammatory steroidal agent is aglucocorticosteroid (synonymously “glucocorticoid”). Variousanti-inflammatory glucocorticoids can be used. These include, by way ofexample and not limitation, natural and synthetic steroidal compoundssuch as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, budesonide, chloroprednisone, ciclesonide, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,contrivazol, deflazacort, desonide, desoximetasone, dexamethansone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flurandrenolone acetonide, flucloronide, flumethasone, flunisolide,fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone,fluorometholone, fluperolone acetate, fluprednidene acetate,fluprednisolone, flurandrenolide, fluticasone propionate, formocortal,halcinode, halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, hydrocortisone 17-butyrate,hydrocortisone 17-valerate, loteprednol etabonate, maziprednone,medrysone, mepredinsone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone21-dimethylaminoacetate, prenisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, and triamcinolonehexacetonide. Other glucocorticosteroids will be apparent to the skilledartisan.

In certain instances, the anti-inflammatory steroid is amineralocorticosteroid (synonymously “mineralocorticoid”). Variousmineralocorticoids include, among others, aldosterone,deoxycorticosterone, deoxycorticosterone acetate, and fludrocortisone.It is to be understood, however, that the characterization of a steroidas a glucocorticosteroid or mineralocorticosteroid are used fordescriptive purposes and is not meant to be exclusionary.Glucocorticoids display some mineralocorticosteroid activity while somemineralocorticoids display some glucocorticoid activity. For thepurposes herein, a mineralocorticoid with anti-inflammatory propertiesmay be used. Generally, mineralocorticosteroids with someglucocorticosteroid activity appears to have anti-inflammatory effects.A certain anti-inflammatory mineralocorticoid is fludrocortisone.

In certain instances, the anti-inflammatory steroidal agents havevarying biologic effect half-life, and can be divided into short acting,intermediate acting, or long acting steroidal compounds. Certainshort-acting steroidal compounds include, by way of example and notlimitation, cortisol and cortisone. Certain intermediate-actingsteroidal compounds include, by way of example and not limitation,prednisone, prednisolone, triamcinolone, and methylprednisolone. Certainlong-acting steroidal compounds include, by way of example and notlimitation, dexamethasone, betamethasone, and budesonide.

In certain instances, the anti-inflammatory steroid is a nitro-steroidalcompound. As used herein a “nitro-steroidal” compound is steroid havingNO-releasing activity (the nitrosterols), and include NO-releasing formsof prednisolone, flunisolide and hydrocortisone.

In certain instances, the steroidal anti-inflammatory agent can be aninhaled steroidal agent, which is useful for nasal administration and/orabsorption through the lungs. These forms are effective agents fortreating asthma and reaction to inhaled allergens. Various forms ofsteroidal anti-inflammatory compounds formulated as inhalants include,among others, beclomethasone, bedesonide, dexamethasone, flunisolide,triamcinolone acetonide, and antedrugs noted above.

In certain instances, the steroidal anti-inflammatory agent is anestrogen or a synthetic estrogen analog. Various estrogen and estrogenanalogs that may be used include, by way of example and not limitation,estrogen, 17β-estradiol, estrogen conjugates, medroxyprogesterone,2-methoxyestradiol (estrogen metabolite), diethystilbesterol,reveratrol, phytoestrogens (e.g., genestein), and tamoxifen.

In certain instances, the steroidal anti-inflammatory compound isvitamin D or an analog thereof. Various anti-inflammatory agents of thisgroup include, by way of example and not limitation,7-dehydrocholesterol, cholecaciferol, ergosterol, 1,25-dihydroxyvitaminD3, and 22-ene-25-oxa-vitamin D. Other vitamin D analogs are describedin U.S. Pat. Nos. 6,924,400; 6,858,595; 6,689,922; and 6,573,256.

Non-Steroidal Anti-inflammatory Agents

In certain instances, the anti-inflammatory agent is a non-steroidalanti-inflammatory agent (NSAID). This class of agents includes aheterogeneous group of compounds with varying structures but which actthrough common therapeutic targets. NSAIDs are classified based on theirchemical structures and biological activities. In certain instances, theNSAIDs useful with the subject compounds are non-selective COX-2inhibitors, which inhibit the activity of both COX-1 and COX-2 isoforms.A certain non-selective COX inhibitor is salicylic acid and derivativesthereof. Certain compounds of this class include, by way of example andnot limitation, acetylsalicylic acid, sodium salicylate, cholinemagnesium trisalicylate, salsalate, diflunisal, sulfasalazine,olsalazine, and mesalamine.

In certain instances, a class of non-selective COX inhibitors is indoleand indene acetic acids. Certain compounds of this class include, amongothers, indomethacin, acemetacin, alclofenac, clidanac, diclofenac,fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac,oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac.

In certain instances, a class of non-selective COX inhibitors isheteroaryl acetic acids. Certain compounds of this class include, amongothers, tolmetin, diclofenac, and ketorolac.

In certain instances, a class of non-selective COX inhibitors isarylpropionic acids or propionic acid derivatives (profens). Certaincompounds of this class include among others, alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen,oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, andtioxaprofen.

In certain instances, a class of non-selective COX inhibitors isanthranilic acids (fenamates). Certain compounds of this class include,among others, flufenamic acid, meclofenamic acid, mefenamic acid,niflumic acid and tolfenamic acid.

In certain instances, a class of non-selective COX inhibitors is enolicacids (e.g., oxicams). Certain compounds of this class include, amongothers, piroxicam and meloxicam, isoxicam, and sudoxicam and tenoxican.

In certain instances, a class of non-selective COX inhibitors isphenylpyrazolones. Certain compounds of this class include, amongothers, phenylbutazone, apazone, bezpiperylon, feprazone, mofebutazone,oxyphenbutazone.

In certain instances, a class of non-selective COX inhibitors isbiphenylcarboxylic acid derivatives. Certain compounds of this classinclude, among others, diflunisal and flufenisal.

In certain instances, the NSAIDs are selective COX-2 inhibitors. As usedherein, a selective COX-2 inhibitor preferably inhibits the activity ofCOX-2 isozyme as compared to the inhibition of the COX-1 isozyme. Aselective COX-2 inhibitor can have a selectivity (i.e., inhibition ofCOX-2/COX-1) of about 10, of about 20 of about 50, of about 100, ofabout 200, of about 500, and of about 1000 or more. Selectivity is basedon assay typically used to measure COX activity.

In certain instances, a class of selective COX-2 inhibitors isdiaryl-substituted furanones. A certain compound of this class includes,among others, refocoxib, available under the tradensme Vioxx™

In certain instances, a class of selective COX-2 inhibitors isdiaryl-substituted pyrazoles. A certain compound of this class includes,among others, celecoxib, available under the tradename Celebrex™

In certain instances, a class of selective COX-2 inhibitors is indoleacetic acids. A certain compound of this class includes, among others,etodolac, available under the tradename Lodine™

In certain instances, a class of selective COX-2 inhibitors issulfonanilides. A certain compound of this class includes, among others,nimesulide.

Lipoxygenase and 5-Lipoxygenase Activating Protein (FLAP) Antagonists

In certain instances, the non-steroidal anti-inflammatory agent that canbe used with the subject compounds is a lipoxygenase or a 5-lipoxygenaseactivating protein (FLAP) antagonist.

In certain instances, various antagonists of lipoxygenase may be used toameliorate the inflammatory response mediated by the leukotrienes.Classes of lipoxygenase inhibitors include, among others, N-hydroxyureaderivatives, redox inhibitors, and non-redox inhibitors. CertainN-hydroxyurea derived inhibitors include, by way of example and notlimitation, 1-(1-benzothiophen-2-ylethyl)-1-hydroxy-urea (leutrol),1-[4-[5-(4-fluorophenoxy)-2-furyl]but-3-yn-2-yl]-1-hydroxy-urea;1-[(2R)-4-[5-[(4-fluorophenyl)methyl]thiophen-2-yl]but-3-yn-2-yl]-1-hydro-xy-urea(atreleuton); 3-(1-benzothiophen-2-ylethyl)-1-hydroxy-urea. A certainredox inhibitor includes, by way of example and not limitation,2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-cyclohexa-2,5-diene-1,4-dione(docebenone). A certain non-redox inhibitor includes, by way of exampleand not limitation,6-[[3-fluoro-5-(4-methoxyoxan-4-yl)phenoxy]methyl]-1-methyl-quinolin-2-one(i.e., ZD2138).

In certain instances, a FLAP antagonist may be used as theanti-inflammatory agent. FLAP antagonists include, among others, indolederivatives and quinoline derivatives. Certain indole derivatives withFLAP inhibitory activity include, by way of example and not limitation,3-[3-butylsulfanyl-1-[(4-chlorophenyl)methyl]-5-propan-2-yl-indol-2-yl]-2-,2-dimethyl-propanoicacid (i.e., MK-866) and3-[1-[(4-chlorophenyl)methyl]-5-(quinolin-2-ylmethoxy)-3-tert-butylsulfan-yl-indol-2-yl]-2,2-dimethyl-propanoicacid (i.e., MK0591 or quiflapon). Certain quinoline derivatives include,by way of example and not limitation,(2R)-2-cyclopentyl-2-[4-(quinolin-2-ylmethoxy)phenyl]acetic acid (i.e.,BAY-X1005 and veliflapon).

Anti-Histamines

In certain instances, the subject compounds are used in combination withanti-histamines, which are generally H1-receptor antagonists. Certain H1receptor antagonists include, among others, doxepin, cabinoxamine,clemastine, diphenylhydramine, dimenhydrinate, pyrilamine,tripelennamine, chlorpheniramine, bromopheniramine, hydroxyzine,cyclizine, meclizine, promethazine, cyproheptadine, phenindamine,acrivastine, citirizine, azelastine, levocabastine, loratadine,fexofenadine, and various salts, hydrates, N-oxides, and prodrugsthereof.

Beta-Agonists

In certain instances, the subject compounds are used in combination withβ-adrenergic receptor agonists (synonymously “β-agonists” orβ-adrenergic agonists”), which includes non-selective β-adrenergicagonists as well as β₂-selective adrenergic agonists. There aregenerally two types of β-agonists, short-acting β-agonists andlong-acting β-adrenergic agonists.

Certain short acting β-adrenergic agonists include, by way of exampleand not limitation, albuterol (salbutamol), isotharine, fenoterol,levalbuterol, metaproterenol (orciprenaline), procaterol, terbutaline,and pirbuterol. Certain long-acting β-adrenergic agonists include, byway of example and not limitation, salmeterol xinafoate, formoterol, andbitolterol. Certain non-selective β-agonists include, by way of exampleand not limitation, isoproterenol and dobutamine.

Anti-Metabolite Anti-Inflammatory Agents

In certain instances, the anti-inflammatory agent is an anti-metabolitethat attenuates or inhibits the activation and/or proliferation of cellsinvolved in inflammation. Anti-metabolites may have cytostatic orcytotoxic effects and thus generally display immunosuppressivecharacteristics.

Various anti-inflammatory anti-metabolites may be used in combinationwith the subject compounds. In certain instances, the anti-proliferativeagent is methotrexate.

In certain instances, the anti-proliferative anti-metabolite includes aninhibitor of inosine monophosphate dehydrogenase (IMPDH), the enzymeacting in the salvage pathway for the synthesis of guanosinemonophosphate (GMP) from inosine. IMPDH inhibitors useful asanti-inflammatory agents include, among others, mycophenolic acid,mycophenolate mofetil, ribavirin, taizofurin, selenazofurin, benazamideadenine dinucleotide, and benzamide riboside.

Other anti-metabolites include azathioprine, 6-mercaptopurine (6-MP),leflunomide, and malononitriloamides.

Another anti-metabolite is methotrexate (amethopterin or(25)-2-[(4-{[(2,4-diamino-7,8-dihydropteridin-6-yl)methyl](methyl)amino}phenyl)formamido]pentanedioicacid).

Anti-TNF-Alpha Agents

It is to be understood that anti-inflammatory agents other than thosedescribed above may be used in combination with the subject compounds.These include various agents directed against the cellular factorsthought to be involved in promoting the inflammatory response. Incertain instances, the anti-inflammatory agent is an agent that blocksthe action of TNFα, the major cytokine implicated in inflammatorydisorders. In certain instances, the anti-TNF is an antibody that blocksthe action of TNFα. A certain anti-TNF antibody is infliximab, availableunder the tradename Remicade®.

In certain instances, the anti-TNFα agent is a receptor construct thatbinds TNFα and prevents its interaction with TNF receptors on present oncells. A certain anti-inflammatory agent based on TNFα receptor isentanercept, available under the tradename Enbrel®.

Statins

In certain instances, the subject compounds are used in combination withstatins. Statins are a class of drugs that can lower cholesterol and actas HMG-CoA reductase inhibitor. Examples of statins includeatorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitastatin, pravastatin, rosuvastatin, and simvastatin. A certain statinis atorvastatin, available under the tradename Lipitor™. Another statinis simvastatin, available under the tradename Zocor®.

Also provided is a method comprising administering a Syk inhibitory2,4-pyrimidinediamine compound and an anti-hypertensive agent to apatient having an inflammatory disorder, thereby treating theinflammatory disorder. In this method, the anti-hypertensive agent maybe selected from the group consisting of: a diuretic, an adrenergicblocker, an angiotensin converting enzyme (ACE) inhibitor, anangiotensin II receptor antagonist, a calcium channel blocker, a directvasodilator, and a neutral endopeptidase inhibitor. Diuretics cause thereduction of water and sodium, or block sodium transport, resulting in areduction in blood pressure. Adrenergic blockers include alpha-blockers,beta-blockers and the alpha/beta blocker labetalol, that block theeffects of the sympathetic nervous system, which responds to stress byraising blood pressure. Angiotensin converting enzyme (ACE) inhibitorslower blood pressure by dilating arteries by blocking the effects of theangiotensin-renin-aldosterone system. Angiotensin II receptorantagonists lower blood pressure by blocking the angiotensin IIreceptor. Calcium channel blockers and direct vasodilators reduce bloodpressure by causing blood vessel dilation. Neutral endopeptidaseinhibitors produce higher levels of atrial natiuretic peptide, whichopens blood vessels. Exemplary anti-hypertensive agents are described inU.S. Patent Application Publications nos. 2006/0160834 and 2007/0092888and are discussed in greater detail below.

In one embodiment, the anti-hypertensive agent may be an ACE inhibitor.ACE inhibitors help relax blood vessels by blocking the cyclization ofangiotensin as well as degrading bradykinin, which both causevasoconstriction. There are different classes of ACE inhibitors. Certainexamples of non-peptide inhibitors chelate zinc and heavy metal ionsneeded for enzymatic activity, and thereby create a catalyticallydefective enzyme. A second class of inhibitors includes peptides andpeptidomimetics that interact with ACE similarly to endogenoussubstrates. Examples of ACE inhibitors include benazepril, captopril,cilazapril, delapril, enalapril, fosinopril, imidapril, losinopril,moexipril, quinapril, quinaprilat, ramipril, perindopril, perindropril,quanipril, spirapril, tenocapril, trandolapril, and zofenopril, andpharmaceutically acceptable salts or esters thereof, suitable dosagesfor which are known to those of skill in the art.

In another embodiment, the anti-hypertensive agent may be an angiotensinII receptor antagonist. Such agents help relax blood vessels by blockingthe angiotensin II receptor (a GPCR), which binds free angiotensin IIand initiates the biochemical signal pathways that lead to manydownstream physiological effects, including vasoconstruction.Candesartan, eprosartan, irbesartan, losartan(2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-methanol),pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K,and RNH6270, and pharmaceutically acceptable salts or esters thereof areexamples of angiotension II receptor antagonists.

In another embodiment, the anti-hypertensive agent may be a calciumchannel blocker. Ca²⁺ acts as an intracellular messenger. Ca²⁺-bindingproteins sense increases in Ca²⁺ concentration and trigger cellularprocesses such as muscle contraction. A calcium channel blocker maytarget CaV1 channels, specifically CaV1.2 channels that are highlyexpressed in cardiac and smooth muscle. The three most commonly employedcalcium channel blocker classes are phenylalkylamines (PAA; e.g.,verapamil), benzothiazepines (e.g., diltiazem), and dihydropyridines(DHP; e.g., nifedipine or amlodipine). Each drug class binds to distinctsites on the α₁ subunit that mediate blocking Examples of calciumchannel blockers include amlodipine, aranidipine, azelnidipine,barnidipine, benidipine, bepridil, cinaldipine, clevidipine, diltiazem,efonidipine, felodipine, gallopamil, isradipine, lacidipine,lemildipine, lercanidipine, nicardipine, nifedipine, nilvadipine,nimodepine, nisoldipine, nitrendipine, manidipine, pranidipine, andverapamil, and pharmaceutically acceptable salts or esters thereof.

In another embodiment, the anti-hypertensive agent is a beta blocker,which agents block sympathetic effects on the heart and are generallyeffective in reducing cardiac output and in lowering arterial pressurewhen there is increased cardiac sympathetic nerve activity. In addition,these agents block the adrenergic nerve-mediated release of renin fromthe renal juxtaglomerular cells. Examples of this group of drugsinclude, but are not limited to, chemical agents such as acebutolol,atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, carteoiol,carvedilol, celiprolol, esmolol, indenolol, metaprolol, nadolol,nebivolol, penbutolol, pindolol, propanolol, sotalol, tertatolol,tilisolol, and timolol, and pharmaceutically acceptable salts or estersthereof. The nonselective beta-blockers, including propranolol,oxprenolol, pindolol, nadolol, timolol and labetalol, which eachantagonize both β₁- and β₂-adrenergic receptors. For the selectiveantagonists, including metoprolol, atenolol, esmolol, and acebutolol,each has much greater binding affinity for the β₁ adrenergic receptor.The selective beta-blockers are normally indicated for patients in whomβ₂-receptor antagonism might be associated with an increased risk ofadverse effects. Such patients include those with asthma or diabetes, orpatients with peripheral vascular disease or Raynaud's disease.

In another embodiment, the anti-hypertensive agent agent is a diuretic,i.e., an agent that affects sodium diuresis and volume depletion in apatent. Diuretic antihypertensives include thiazides (such ashydrochlorothiazide, chlorothiazide, and chlorthalidone), metolazone,loop diuretics (such as furosemide, bumetanide, ethacrynic acid,piretanide and torsemide), and aldosterone antagonists (such asspironolactone, triamterene, and amiloride).

Other anti-hypertensive agents suitable for use in combination with thepresently disclosed compounds include renin inhibitors (e.g., aliskirenand tekturna, which slow down the production of renin), alpha-blockers(e.g, alpha 2a agonists such as lofexidine, tiamenidine, moxonidine,rilmenidine and guanobenz and alpha 1 blockers such as terazosin,urapidil, prazosin, bunazosin, trimazosin, doxazosin, naftopidil,indoramin, WHIP 164, and XEN010, and pharmaceutically acceptable saltsor esters thereof), alpha-beta blockers (e.g., nipradilol, arotinololand amosulalol, and pharmaceutically acceptable salts or estersthereof), central-acting agents (which prevent the brain from signalingthe nervous system to increase heart rate and narrow blood vessels),vasodilators, e.g., hydralazine (apresoline), clonidine (catapres),minoxidil (loniten), and nicotinyl alcohol (roniacol); andpharmaceutically acceptable salts or esters thereof) and endothelinantagonists (e.g., tezosentan, A308165, and YM62899, andpharmaceutically acceptable salts or esters thereof).

In particular, when the presently disclosed compounds are used to treata cell proliferative disorder, they may be administered in combinationwith one or more therapeutically or prophylactically effectivechemotherapeutic agents. Suitable chemotherapeutic agents for use incombination with the presently disclosed compounds include, by way ofexample, those generally described as angiogenesis inhibitors,antimetabolites, alkylating agents, coordination compounds, platinumcomplexes, DNA cross-linking compounds, inhibitors of transcriptionenzymes, protein kinase inhibitors, including tyrosine kinaseinhibitors, topoisomerase inhibitors, DNA minor-groove bindingcompounds, vinca alkyloids, taxanes, antitumor antibiotics, hormones,aromatase inhibitors, enzymes, growth factor receptors antibodies,cytokines, cell surface markers antibodies, HDAC inhibitors, HSP 90inhibitors, BCL-2 inhibitors, mTOR inhibitors, proteasome inhibitors ormonoclonal antibodies.

Accordingly, one embodiment of methods of preventing, treating ormanaging cancer in a subject in need thereof comprises administering tothe subject a therapeutically or prophylactically effective amount ofpresently disclosed compound in combination with the administration of atherapeutically or prophylactically effective amount of one or morechemotherapeutic agents, wherein one or more chemotherapeutic agents areindependently selected from the group consisting of mechlorothamine,cyclophosphamide, ifosfamide, melphalan, chlorambucil, ethyleneimines,methylmelamines, procarbazine, dacarbazine, temozolomide, busulfan,carmustine, lomustine, methotrexate, fluorouracil, capecitabine,cytarabine, gemcitabine, cytosine arabinoside, mercaptopurine,fludarabine, cladribine, thioguanine, azathioprine, vinblastine,vincristine, paclitaxel, docetaxel, colchicine, actinomycin D,daunorubicin, bleomycin,L-asparaginase, cisplatin, carboplatin,oxaliplatin, prednisone, dexamethasone, amino glutethimide, formestane,anastrozole, hydroxyprogesterone caproate, medroxyprogesterone,tamoxifen, amsacrine, mitoxantrone, topotecan, irinotecan, camptothecin,axtinib, bosutinib, carfilzomib, cediranib, dasatinib, erlotinib,gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib,sunitinib, vandetanib, vatalanib, anti-Her2 antibodies, interferon-α,interferon-γ, interleukin-2, GM-CSF, anti-CTLA-4 antibodies, rituximab,anti-CD33 antibodies, MGCD0103, vorinostat, 17-AAG, thalidomide,lenalidomide, rapamycin, CCI-779, sorafenib, doxorubicine, gemcitabine,melphalan, bortezomib, NPI052, gemtuzumab, alemtuzumab, ibritumomabtiuxaetan, tositumomab, iodine-131 tositumomab, trastuzumab,bevacizumab, rituximab, and anti-TRAIL death receptor antibodies.

Pharmaceutical Compositions

In another aspect, the present disclosure provides compositionscomprising one or more of compounds or salts as described above withrespect to any of formulae (I)-(III) and an appropriate carrier,excipient or diluent. The exact nature of the carrier, excipient ordiluent will depend upon the desired use for the composition, and mayrange from being suitable or acceptable for veterinary uses to beingsuitable or acceptable for human use. The composition may optionallyinclude one or more additional compounds.

When used to treat or prevent such diseases, the compounds describedherein may be administered singly, as mixtures of one or more compoundsor in mixture or combination with other agents useful for treating suchdiseases and/or the symptoms associated with such diseases. Thecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5L0 inhibitors, leukotriene synthesis and receptorinhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgGisotype switching or IgG synthesis, β-agonists, tryptase inhibitors,aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to namea few. The compounds may be administered in the form of compounds perse, or as pharmaceutical compositions comprising a compound.

Pharmaceutical compositions comprising the compound(s) may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making levigating, emulsifying, encapsulating, entrapping orlyophilization processes. The compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the compounds into preparations which can be usedpharmaceutically.

The compounds may be formulated in the pharmaceutical composition perse, or in the form of a hydrate, solvate, N-oxide or pharmaceuticallyacceptable salt, as previously described. Typically, such salts are moresoluble in aqueous solutions than the corresponding free acids andbases, but salts having lower solubility than the corresponding freeacids and bases may also be formed.

Pharmaceutical compositions may take a form suitable for virtually anymode of administration, including, for example, topical, ocular, oral,buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc.,or a form suitable for administration by inhalation or insufflation.

For topical administration, the compound(s) may be formulated assolutions, gels, ointments, creams, suspensions, etc. as are well-knownin the art. Systemic formulations include those designed foradministration by injection, e.g., subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal oral or pulmonaryadministration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives. Alternatively, theinjectable formulation may be provided in powder form for reconstitutionwith a suitable vehicle, including but not limited to sterile pyrogenfree water, buffer, dextrose solution, etc., before use. To this end,the active compound(s) may be dried by any art-known technique, such aslyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets may be coated by methods well known in theart with, for example, sugars, films or enteric coatings.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™ or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the compound, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the compound(s) may beformulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For nasal administration or administration by inhalation orinsufflation, the compound(s) can be conveniently delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbondioxide or other suitable gas. In the case of a pressurized aerosol, thedosage unit may be determined by providing a valve to deliver a meteredamount. Capsules and cartridges for use in an inhaler or insufflator(for example capsules and cartridges comprised of gelatin) may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

For ocular administration, the compound(s) may be formulated as asolution, emulsion, suspension, etc. suitable for administration to theeye. A variety of vehicles suitable for administering compounds to theeye are known in the art.

For prolonged delivery, the compound(s) can be formulated as a depotpreparation for administration by implantation or intramuscularinjection. The compound(s) may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, e.g., as asparingly soluble salt. Alternatively, transdermal delivery systemsmanufactured as an adhesive disc or patch which slowly releases thecompound(s) for percutaneous absorption may be used. To this end,permeation enhancers may be used to facilitate transdermal penetrationof the compound(s).

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver compound(s). Certain organic solvents suchas dimethylsulfoxide (DMSO) may also be employed, although usually atthe cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the compound(s). The pack may, for example, comprise metal orplastic foil, such as a blister pack. The pack or dispenser device maybe accompanied by instructions for administration.

The compound(s) described herein, or compositions thereof, willgenerally be used in an amount effective to achieve the intended result,for example in an amount effective to treat or prevent the particulardisease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit or prophylactically toachieve prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treatedand/or eradication or amelioration of one or more of the symptomsassociated with the underlying disorder such that the patient reports animprovement in feeling or condition, notwithstanding that the patientmay still be afflicted with the underlying disorder. For example,administration of a compound to a patient suffering from an allergyprovides therapeutic benefit not only when the underlying allergicresponse is eradicated or ameliorated, but also when the patient reportsa decrease in the severity or duration of the symptoms associated withthe allergy following exposure to the allergen. As another example,therapeutic benefit in the context of asthma includes an improvement inrespiration following the onset of an asthmatic attack, or a reductionin the frequency or severity of asthmatic episodes. Therapeutic benefitin the context of rheumatoid arthritis also includes the ACR20, or ACR50or ACR70, as previously described. Therapeutic benefit also generallyincludes halting or slowing the progression of the disease, regardlessof whether improvement is realized.

For prophylactic administration, the compound(s) may be administered toa patient at risk of developing one of the previously describeddiseases. For example, if it is unknown whether a patient is allergic toa particular drug, the compound(s) may be administered prior toadministration of the drug to avoid or ameliorate an allergic responseto the drug. Alternatively, prophylactic administration may be appliedto avoid the onset of symptoms in a patient diagnosed with theunderlying disorder. For example, the compound(s) may be administered toan allergy sufferer prior to expected exposure to the allergen.Compound(s) may also be administered prophylactically to healthyindividuals who are repeatedly exposed to agents known to one of theabove-described maladies to prevent the onset of the disorder. Forexample, compound(s) may be administered to a healthy individual who isrepeatedly exposed to an allergen known to induce allergies, such aslatex, in an effort to prevent the individual from developing anallergy. Alternatively, compound(s) may be administered to a patientsuffering from asthma prior to partaking in activities which triggerasthma attacks to lessen the severity of, or avoid altogether, anasthmatic episode.

The amount of compound(s) administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular compound(s) the conversation rate and efficiency intoactive drug compound under the selected route of administration, etc.

Determination of an effective dosage of compound(s) for a particular useand mode of administration is well within the capabilities of thoseskilled in the art. Effective dosages may be estimated initially from invitro activity and metabolism assays. For example, an initial dosage ofcompound for use in animals may be formulated to achieve a circulatingblood or serum concentration of the metabolite active compound that isat or above an IC₅₀ of the particular compound as measured in as invitro assay. Calculating dosages to achieve such circulating blood orserum concentrations taking into account the bioavailability of theparticular compound via the desired route of administration is wellwithin the capabilities of skilled artisans. Initial dosages of compoundcan also be estimated from in vivo data, such as animal models. Animalmodels useful for testing the efficacy of the active metabolites totreat or prevent the various diseases described above are well-known inthe art. Animal models suitable for testing the bioavailability and/ormetabolism of compounds into active metabolites are also well-known.Ordinarily skilled artisans can routinely adapt such information todetermine dosages of particular compounds suitable for humanadministration.

Dosage amounts will typically be in the range of from about 0.0001mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, butmay be higher or lower, depending upon, among other factors, theactivity of the active metabolite compound, the bioavailability of thecompound, its metabolism kinetics and other pharmacokinetic properties,the mode of administration and various other factors, discussed above.For example, a therapeutically effective dosage may be from about 10 mgper day to about 600 mg per day, such as from about 20 mg per day toabout 400 mg per day and in particular from about 75 mg to about 300 mgper day. In a particular dosage regimen a subject receives 75 mg, 100 mgor 150 mg once or twice daily. Dosage amount and interval may beadjusted individually to provide plasma levels of the compound(s) and/oractive metabolite compound(s) which are sufficient to maintaintherapeutic or prophylactic effect. For example, the compounds may beadministered once per week, several times per week (e.g., every otherday), once per day or multiple times per day, depending upon, amongother things, the mode of administration, the specific indication beingtreated and the judgment of the prescribing physician. In cases of localadministration or selective uptake, such as local topicaladministration, the effective local concentration of compound(s) and/oractive metabolite compound(s) may not be related to plasmaconcentration. Skilled artisans will be able to optimize effective localdosages without undue experimentation.

DEFINITIONS

As used herein, the following terms are intended to have the followingmeanings:

The term “isotopic enrichment factor” as used herein means the ratiobetween the isotopic abundance and the natural abundance of a specifiedisotope. It will be recognized that some variation of natural isotopicabundance occurs in a synthesized compound depending upon the origin ofchemical materials used in the synthesis. Thus, a preparation of anycompound will inherently contain small amounts of deuteratedisotopologues. The concentration of naturally abundant stable hydrogenisotopes, notwithstanding this variation, is small and immaterial ascompared to the degree of stable isotopic substitution of compounds ofthis disclosure. In a compound of this disclosure, when a particularposition is designated as having deuterium, it is understood that theabundance of deuterium at that position is substantially greater thanthe natural abundance of deuterium, which is about 0.015% (on a mol/molbasis). A position designated as having deuterium will often have aminimum isotopic enrichment factor of at least 3000 (45% deuteriumincorporation) at each atom designated as deuterium in the compound.

In the compounds of this disclosure any atom not specifically designatedas a particular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at about its natural abundance isotopic composition.

The term “isotopologue” refers to a species that has the same chemicalstructure and formula as another compound, with the exception of theisotopic composition at one or more positions, e.g., H vs. D. Thusisotopologues differ in their isotopic composition.

“Fc Receptor” refers to a member of the family of cell surface moleculesthat binds the Fc portion (containing the specific constant region) ofan immunoglobulin. Each Fc receptor binds immunoglobulins of a specifictype. For example the Fcα receptor (“FcαR”) binds IgA, the FcεR bindsIgE and the FcγR binds IgG.

The FcαR family includes the polymeric Ig receptor involved inepithelial transport of IgA/IgM, the myeloid specific receptor ReαRI(also called CD89), the Fcα/μR and at least two alternative IgAreceptors (for a recent review see Monteiro & van de Winkel, 2003, AnnuRev. Immunol, advanced e-publication). The FcαRI is expressed onneutrophils, eosinophils, monocytes/macrophages, dendritic cells andkupfer cells. The FcαRI includes one alpha chain and the FcR gammahomodimer that bears an activation motif (ITAM) in the cytoplasmicdomain and phosphorylates Syk kinase.

The FcεR family includes two types, designated FcεRI and FcεRII (alsoknown as CD23). FcεRI is a high affinity receptor (binds IgE with anaffinity of about 10¹⁰M⁻¹) found on mast, basophil and eosinophil cellsthat anchors monomeric IgE to the cell surface. The FcεRI possesses onealpha chain, one beta chain and the gamma chain homodimer discussedabove. The FcεRII is a low affinity receptor expressed on mononuclearphagocytes, B lymphocytes, eosinophils and platelets. The FcεRIIcomprises a single polypeptide chain and does not include the gammachain homodimer.

The FcγR family includes three types, designated FcγRI (also known asCD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16).FcγRI is a high affinity receptor (binds IgG1 with an affinity of 10⁸M⁻¹) found on mast, basophil, mononuclear, neutrophil, eosinophil,dendritic and phagocyte cells that anchors nomomeric IgG to the cellsurface. The FcγRI includes one alpha chain and the gamma chain dimershared by FcαRI and FcεRI.

The FcγRII is a low affinity receptor expressed on neutrophils,monocytes, eosinophils, platelets and B lymphocytes. The FcγRII includesone alpha chain, and does not include the gamma chain homodimerdiscussed above.

The FcγRIII is a low affinity (binds IgG1 with an affinity of 5×10⁵ M⁻¹)expressed on NK, eosinophil, macrophage, neutrophil and mast cells. Itcomprises one alpha chain and the gamma homodimer shared by FcαRI, FcεRIand FcγRI.

Skilled artisans will recognize that the subunit structure and bindingproperties of these various Fc receptors, as well as the cell typesexpressing them, are not completely characterized. The above discussionmerely reflects the current state-of-the-art regarding these receptors(see, e.g., Immunobiology: The Immune System in Health & Disease, 5^(th)Edition, Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, FIG. 9. 30 atpp. 371), and is not intended to be limiting with respect to the myriadreceptor signaling cascades that can be regulated with the compoundsdescribed herein.

“Fc Receptor-Mediated Degranulation” or “Fc Receptor-InducedDegranulation” refers to degranulation that proceeds via an Fc receptorsignal transduction cascade initiated by crosslinking of an Fc receptor.

“IgE-Induced Degranulation” or “FcεRI-Mediated Degranulation” refers todegranulation that proceeds via the IgE receptor signal transductioncascade initiated by crosslinking of FcεR1-bound IgE. The crosslinkingmay be induced by an IgE-specific allergen or other multivalent bindingagent, such as an anti-IgE antibody. In mast and/or basophil cells, theFcεRI signaling cascade leading to degranulation may be broken into twostages: upstream and downstream. The upstream stage includes all of theprocesses that occur prior to calcium ion mobilization. The downstreamstage includes calcium ion mobilization and all processes downstreamthereof. Compounds that inhibit FcεRI-mediated degranulation may act atany point along the FcεRI-mediated signal transduction cascade.Compounds that selectively inhibit upstream FcεRI-mediated degranulationact to inhibit that portion of the FccRI signaling cascade upstream ofthe point at which calcium ion mobilization is induced. In cell-basedassays, compounds that selectively inhibit upstream FcεRI-mediateddegranulation inhibit degranulation of cells such as mast or basophilcells that are activated or stimulated with an IgE-specific allergen orbinding agent (such as an anti-IgE antibody) but do not appreciablyinhibit degranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcεRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“IgG-Induced Degranulation” or “FcγRI-Mediated Degranulation” refers todegranulation that proceeds via the FcγRI signal transduction cascadeinitiated by crosslinking of FcγRI-bound IgG. The crosslinking may beinduced by an IgG-specific allergen or another multivalent bindingagent, such as an anti-IgG or fragment antibody. Like the FcεRIsignaling cascade, in mast and basophil cells the FcγRI signalingcascade also leads to degranulation which may be broken into the sametwo stages: upstream and downstream. Similar to FcεRI-mediateddegranulation, compounds that selectively inhibit upstreamFcγRI-mediated degranulation act upstream of the point at which calciumion mobilization is induced. In cell-based assays, compounds thatselectively inhibit upstream FcγRI-mediated degranulation inhibitdegranulation of cells such as mast or basophil cells that are activatedor stimulated with an IgG-specific allergen or binding agent (such as ananti-IgG antibody or fragment) but do not appreciably inhibitdegranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcγRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“Ionophore-Induced Degranulation” or “Ionophore-Mediated Degranulation”refers to degranulation of a cell, such as a mast or basophil cell, thatoccurs upon exposure to a calcium ionophore such as, for example,ionomycin or A23187.

“Syk Kinase” refers to the well-known 72 kDa non-receptor (cytoplasmic)spleen protein tyrosine kinase expressed in B-cells and otherhematopoetic cells. Syk kinase includes two consensus Src-homology 2(SH2) domains in tandem that bind to phosphorylated immunoreceptortyrosine-based activation motifs (“ITAMs”), a “linker” domain and acatalytic domain (for a review of the structure and function of Sykkinase see Sada et al., 2001, J. Biochem. (Tokyo) 130:177-186); see alsoTurner et al., 2000, Immunology Today 21:148-154). Syk kinase has beenextensively studied as an effector of B-cell receptor (BCR) signaling(Turner et al., 2000, supra). Syk kinase is also critical for tyrosinephosphorylation of multiple proteins which regulate important pathwaysleading from immunoreceptors, such as Ca²⁺ mobilization andmitogen-activated protein kinase (MAPK) cascades and degranulation. Sykkinase also plays a critical role in integrin signaling in neutrophils(see, e.g., Mocsai et al. 2002, Immunity 16:547-558).

As used herein, Syk kinase includes kinases from any species of animal,including but not limited to, homosapiens, simian, bovine, porcine,rodent, etc., recognized as belonging to the Syk family. Specificallyincluded are isoforms, splice variants, allelic variants, mutants, bothnaturally occurring and man-made. The amino acid sequences of such Sykkinases are well known and available from GENBANK. Specific examples ofmRNAs encoding different isoforms of human Syk kinase can be found atGENBANK accession no. gi|21361552|ref|NM_(—)003177.2|,gi|496899|emb|Z29630.1|HSSYKPTK[496899] andgi|15030258|gb|BC011399.1|BC011399[15030258], which are incorporatedherein by reference.

Skilled artisans will appreciate that tyrosine kinases belonging toother families may have active sites or binding pockets that are similarin three-dimensional structure to that of Syk. As a consequence of thisstructural similarity, such kinases, referred to herein as “Syk mimics,”are expected to catalyze phosphorylation of substrates phosphorylated bySyk. Thus, it will be appreciated that such Syk mimics, signaltransduction cascades in which such Syk mimics play a role, andbiological responses effected by such Syk mimics and Syk mimic-dependentsignaling cascades may be regulated, and in particular inhibited, withmany of the compounds described herein.

“Syk-Dependent Signaling Cascade” refers to a signal transductioncascade in which Syk kinase plays a role. Non-limiting examples of suchSyk-dependent signaling cascades include the FcαRI, FcεRI, FcγRI,FcγRIII, BCR and integrin signaling cascades.

“Autoimmune Disease” refers to those diseases which are commonlyassociated with the nonanaphylactic hypersensitivity reactions (Type II,Type III and/or Type IV hypersensitivity reactions) that generallyresult as a consequence of the subject's own humoral and/orcell-mediated immune response to one or more immunogenic substances ofendogenous and/or exogenous origin. Such autoimmune diseases aredistinguished from diseases associated with the anaphylactic (Type I orIgE-mediated) hypersensitivity reactions.

Methods of Synthesis

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via silica gel and/or aluminachromatography. See, e.g., Introduction to Modern Liquid Chromatography,2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons,1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, NewYork, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4.sup.th edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

Those of skill in the art of organic synthesis will find examplessuitable for synthesizing the present compounds in U.S. Pat. Nos.7,122,542, 7,449,458, 7,517,886 and 7,557,210, and in internationalpatent application no. PCT/US03/03022 filed Jan. 31, 2003 (published asWO 03/063794), international patent application no. PCT/US07/85313 filedNov. 20, 2007 (published as WO 2008/064274), and international patentapplication no. PCT/US06/01945 filed Jan. 19, 2006 (published as WO2006/078846). Each of these patents and publications is herebyincorporated herein by reference in its entirety.

EXAMPLES

The compounds of the disclosure are illustrated further by the followingexamples, which are provided for illustrative purposes and are notintended to be construed as limiting the disclosure in scope or spiritto the specific compounds described in them.

Example 1N4-(2,2-Dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-[6-morpholinopyridin-3-yl]-2,4-pyrimidinediamine(Compound 1)

¹H NMR (DMSO-d₆): 8.43 (d, 1H, J=2.0 Hz), 8.15 (d, 1H, J=3.5 Hz), 8.06(d, 1H, J=9.1 Hz), 7.44 (m, 2H), 7.14 (m, 1H), 3.71 (m, 4H), 3.48 (m,4H), 1.41 (s, 6H); LCMS: purity: 99%; MS (m/e): 467 (MH⁺).

Example 2 N4-[2,2-Dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl]-5-fluoro-N2-(6-morpholinopyridin-3-yl)-2,4-pyrimidinediaminedisodium salt (Compound 2)

¹H NMR (D₂O): δ 7.71 (d, 1H, 2.3 Hz), 7.52 (d, 1H, J=4.1 Hz), 7.27 (dd,1H, J=2.3 and 9.1 Hz), 7.17 (d, 1H, J=8.8 Hz), 6.67 (d, 1H, J=8.8 Hz),6.33 (d, 1H, J=9.1 Hz), 5.43 (d, 1H, J=2.3 Hz), 3.64 (s, 4H), 3.02 (s,4H), 1.25 (s, 6H); LCMS: purity: 99%; MS (m/e): 577 (MH⁺-2Na+2H).

Example 3N4-(2,2-Dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(4-morpholinophenyl)-2,4-pyrimidinediamine(Compound 3)

¹H NMR (DMSO-d₆): δ 1.43 (s, 6H), 2.99 (t, J=4.8 Hz, 4H), 3.72 (t, J=4.8Hz, 4H), 6.80 (d, J=8.7 Hz, 2H), 7.36 (d, J=8.4 Hz, 1H), 7.46 (d, J=9.0Hz, 2H), 7.55 (d, J=8.7 Hz, 1H), 8.06 (d, J=3.6 Hz, 1H), 9.00 (s, 1H),9.13 (s, 1H), 11.09 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆): δ −173.16;LCMS: ret. time: 9.59 min.; purity: 100%; MS (m/e): 466.28 (MH⁺).

Example 4 N4-[2,2-Dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrido[1,4]oxazin-6-yl]-5-fluoro-N2-(4-morpholinophenyl)-2,4-pyrimidinediaminedisodium salt (Compound 4)

¹H NMR (D₂O): δ 7.60 (d, 1H, J=4.1 Hz), 7.38 (d, 1H, J=8.8 Hz), 6.99 (d,2H, J=9.1 Hz), 6.78 (d, 1H, J=8.8 Hz, 1H), 6.66 (d, 2H, J=9.1 Hz), 5.45(d, 2H, J=2.3 Hz), 3.71 (app s, 4H), 2.87 (app s, 4H), 1.29 (s, 6H).LCMS: purity: 99%; MS (m/e): 576 (MH+−2Na+2H).

Tryptase Release Assay of Exemplary Compounds

Compounds are assayed for inhibition of mast cell activation induced byFcγR cross-linking by measuring the activity of tryptase released upondegranulation as follows:

Human mast cells are cultured and differentiated from CD38-negativeprogenitor cell as described in U.S. Patent Publication No.2005/0234049, which is hereby incorporated herein by reference in itsentirety. For example, 65 μL of various concentrations of the testcompound are prepared in MT (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.0mM MgCl₂, 5.6 mM Glucose, 20 mM Hepes (pH 7.4), 0.1% Bovine SerumAlbumin, (Sigma, #A4503)) containing 2% MeOH and 1% DMSO, or controlbuffer are added to duplicate 96-well V-bottom plates. Pelleted andre-suspended (in warm MT) CHMC cells (65 μL) are added to each 96-wellplate, mixed and incubated for 1 hour at 37° C. 25 μL of 6× anti-IgGRabbit anti-human IgG, affinity purified (Bethyl Laboratories,#A80-105A3), final concentration 1 μg/mL, is added to the test wells. MT(25 μL) is added to control wells. After a 60-minute incubation at 37°C., cells and cell debris were pelleted by centrifugation at 1000 rpmfor 10 min and tryptase and leukotriene C₄ levels are measured.

To measure tryptase levels, 25 μL of supernatant from each well istransferred to a fresh 96-well black bottom plate, to which 100 μL offresh tryptase substrate solution [(Z-Ala-Lys-Arg-AMC2TFA; EnzymeSystems Products, #AMC-246)]1:2000 in tryptase assay buffer [0.1M Hepes(pH 7.5), 10% w/v Glycerol, 10 μM Heparin (Sigma H-4898) 0.01% NaN₃] isadded. After 30 minutes incubation at room temperature, the opticaldensity of the plates is measured at 355 nm/460 nm on aspectrophotometric plate reader.

Compounds of the disclosure were assayed for their ability to inhibitmast cell activation induced by FcγR cross-linking by measuring theactivity of tryptase released upon degranulation. The IC₅₀ values forthe LD Tryptase assay are presented in Table 1.

TABLE 1 IC₅₀ (LD Tryptase Compound No. CHMC) (μM) Compound 1 0.09Compound 2 21.90 Compound 3 0.08 Compound 4 2.36

While Compound 2 exhibits only micromolar inhibition, it will metabolizeto Compound 1, which has an IC₅₀ value less than 100 nm. Similarly,while Compound 4 exhibits only micromolar inhibition, it will metabolizeto Compound 3, which has an IC₅₀ value less than 100 nm.

Fluorescence Polarization Syk kinase assay

Compounds are tested for the ability to inhibit Syk kinase catalyzedphosphorylation of a peptide substrate in a biochemical fluorescencepolarization assay with isolated Syk kinase.

Test compound stock solutions (10 mM) containing are serially diluted inDMSO starting from 2.5 mM and then further diluted to desiredconcentration of 50 μM (5×) to yield 2% DMSO concentration in kinasebuffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mLacetylated Bovine Gamma Globulin). The assay is carried out in a black96 well low volume plate (Molecular Devices, #42-000-0117) bytransferring compound in 2% DMSO (0.4% DMSO final) pre-mixed withATP/substrate (TK2 peptide) in kinase buffer at room temperature. Sykkinase (Millipore, #14-314) is added to a final reaction volume of 20μL, and the reaction is incubated for 30 minutes at room temperature.Final enzyme reaction conditions are 20 mM HEPES, pH 7.4, 5 mM MgCl₂, 2mM MnCl₂, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin(Invitrogen, #P2255), 25 ng Syk, 2.5 μM ATP, 5 μM peptide substrate(Biotin-EGPWLEEEEEAYGWMDF-CONH₂, Anaspec, #60329-1). The reaction isstopped by addition of 20 μL of PTK quench mix containing EDTA (10 mMfinal)/anti-phosphotyrosine antibody (1× final)/fluorescentphosphopeptide tracer (0.5× final) diluted in FP dilution buffer to stopthe reaction to a total volume of 40 μL according to the manufacturer'sinstructions (Invitrogen). The plate is incubated in the dark foradditional 30 minutes at room temperature and then read on a Polarionfluorescence polarization plate reader (Tecan). Data are converted toamount of phosphopeptide present using a calibration curve generated bycompetition with the phosphopeptide competitor provided in the TyrosineKinase Assay Kit, Green (Invitrogen, #P2837).

CD63 Assay

Compounds are tested for the ability to inhibit allergen inducedbasophil degranulation. The BASOTEST® kit (Orpegen Pharma GmbH,#10-0500) is used in this assay. In a 5 mL FACS tube 105 μL ofHeparinized whole blood, 25 μL of test compound solution (in DMSO) areincubated at room temperature for 30-60 minutes. To inducedegranulation, 20 μL stimulation buffer (Reagent B) is added, and thetube is incubated for 10 min in a 37° C. water bath. Then, 100 μL ofanti-IgE (2 μg/mL) is added, or 100 L of washing solution (reagent A) isadded (i.e., as a negative control). The tube is incubated for 20 min ina 37° C. water bath. Degranulation is then stopped by incubation on icefor 5 min. 20 μL of staining reagent (reagent F) is added, and the tubeis incubated on ice in the dark for 20 min. 2 mL of room temperaturelysing solution is added, and the tube is incubated at room temperaturefor 10 min. The tubes are spun down at 4° C. for 5 min at 1600 rpm, andthe supernatant is discarded. 3 mL washing solution (reagent A) isadded, and the tubes spun down at 4° C. for 5 min at 1600 rpm, and thesupernatant is discarded. 200 μL washing solution is added to theremaining cell pellet, and the samples are incubated on ice in the darkuntil analysis (within 2 h). Analysis is performed using flow cytometryat 488 nm using the conditions specified in the BASOTEST® kitinstructions to determine percentage of activated basophilicgranulocytes.

Biochemical VEGFR Assay

Compounds are tested for the ability to inhibit VEGF2 in an ELISA assay.NUNC MAXISORP 96 well plates (#436110) are coated with 0.01 mg/mLNeutrAvidin in 1×PBS (100 μL/well) for 18-24 h at 4° C. Plates are thenwashed with 1×PBST using a plate washer, then blocked with 2% BSA in1×PBST (100 μL/well) for 1 hr at room temperature. TheNeutrAvidin-coated plates are again washed with 1×PBST using a platewasher.

Test compound solutions (4.8 μL/well) of various concentrations (inDMSO) are transferred to wells of a fresh uncoated 96-well plate, alongwith 115 μL/well of reaction solution (4700 parts kinase buffer (5772parts water, 120 parts 1M HEPES (pH 7.4), 30 parts 1M MgCl₂, 12 parts 1MMnCl₂, 6 parts 1M DTT, 60 parts 1% Brij-35), 1.14 parts 10 mM ATP, 11.4parts 1 mM TK2 peptide (AnaSpec, #60329-1)). The mixed testcompound/reaction solutions are added to the wells of theNeutrAvidin-coated plates (50 μL/well). 6× enzyme solution (1000 partskinase buffer, 0.6 parts KDR/VEGFR2 enzyme (50 μg/mL, Millipore,#14-630) is added to all wells except those designated as negativecontrols. The plates are incubated for 30 minutes on a shaker at roomtemperature.

Detection reagent is prepared by mixing 10000 parts 0.1% BSA in PBSTwith 1 part anti-pTyr mouse mAb (Cell Signaling, #9411) and 1 partHRP-goat anti-mouse IgG (Jackson Immunoresearch, #115-035-003). Thedetection reagent is added at 100 μL/well, and the plates are incubatedfor 60 minutes at room temperature, then washed with 1×PBST using aplate washer. Plates are developed by adding 100 L of ELISA Pico Chemisubstrate (Fisher Scientific, #PI-37069), and read by chemiluminescence(0.1 s) using a Wallac 1420 counter.

Cellular VEGFR Assay

Compounds are tested for the ability to exhibit VEGF-inducedphosphorylation of VEGFR in HUVEC cells.

96-well high binding opaque white plates (Pierce, #15042) are coatedovernight with 5 μg/mL of mouse anti-human VEGFR2 mAb (R&D Systems,#MAB3572). The coated plates are then blocked with 2% BSA in PBS for 2hours.

HUVEC cells (Cambrex, #CC-2519, cultured in complete medium andincubated in a humidified atmosphere of 5% CO₂ at 37° C.) are seeded ata density of 18-20K cells/well in 100 μL, complete medium (EGM-2BulletKit, Cambrex, #CC-3162) in a 96-well clear bottom plate for 24hours in a 37° C./5% CO₂ incubator. Cells are washed 1× with PBS. 100 μLstarvation medium (EBM2 media, Cambrex, #CC3156, containing 1% BSA and0.2% FBS) is added and cells are returned to the incubator for 20-24hours. Test compounds or control drugs are serially diluted in DMSO in acompound plate and further diluted 1:250 in starvation medium. 100 μL,of this 2× concentration is dosed to the cells. After a 1 hourpre-treatment, the cells are stimulated with 100 ng/mL recombinant humanVEGF165 (R&D Systems, #293-VE) for 5 minutes. Immediately afterstimulation, the cells are washed 1× with cold PBS and 33 μL, cold lysisbuffer (9 ml RIPA buffer, Teknova, #R3792) containing a proteaseinhibitor tablet (Roche, #1697498), 1 mM NEM, 1 mM PMSF, 10 μM MG132,and 1 mM NaVO₄ and 1 mL 10× cell lysis buffer (Cell Signal Technology,#9803) is added. Plates are then placed on a rocker/shaker at 4° C. for1 hour.

Phosphorylated VEGFR2 is determined by ELISA. After a wash with TBST, 30μL/well of the cell lysate is transferred into the anti-human VEGFR2mAb-coated plates (described above) containing 200 μL/well of 1% BSA inPBS, and incubated with shaking at 4° C. overnight. The plates arewashed 4× with TBST and stained with 1:1000 diluted phospho-VEGFR2Rabbit mAb (Cell Signal Technology, 2478) in 0.2% BSA in TBST, andallowed to shake for 2 hours. Plates are washed 4× with TBST, and 1:5000dilution anti-Rabbit HRP (Jackson ImmunoResearch, #111-035-144) in 0.2%BSA in TBST is added and the plates are placed on the shaker for anotherhour. A final 4× wash with TBST and SuperSignal ELISA Pico Substrate(Pierce, #37070a/b) was added at a 1:1:1 ratio of test compound, A, Band water for chemiluminescent detection of the HRP conjugate. Thesignal is read using a SpectraMax M5 plate reader.

Cellular Ret Assay

Compounds are tested for the ability to inhibit Ret kinase in cells.SK-N-SH brain neuroblastoma cells (ATCC, #HTB-1, maintained and platedin DMEM (Cellgro Mediatech, #10-013-CV) with 10% fetal bovine serum(JRH, #12106-500M) are seeded in 10 cm plates in 10 mL of culture mediaand allowed to reach 85% confluence by the next day. The medium isreplaced with 5 mL of DMEM (without fetal bovine serum) containing DMSOor test compound (final 0.1% DMSO), and incubated at 37° C./5% CO₂ for 1hour. SK-N-SH cells are then stimulated with 50 ng/mL of humanrecombinant GDNF (Peprotech, #450-10) for 10 minutes. Cells are washedonce with cold 1×PBS and lysed with 500 μL of 1% NP-40 lysis buffer(Tris HCl pH 7.4 with 150 mM NaCl, 1 mM NaVn, 1% Nonidet-P40 (FisherScientific, #PI-28324), protease inhibitor tablet (Roche, 1697498)).Cells are scraped off the plate in lysis buffer after sitting on ice for10 minutes. The detergent-insoluble fraction is removed bycentrifugation at 14,000 rpm for 10 minutes at 4° C. Ret isimmunoprecipitated from the detergent-soluble cell lysate by rotationwith 3 μL of anti-Ret rabbit polyclonal antibody (Cell SignalingTechnology, Cat# 3220) and 15 μL of protein A/G agarose (FisherScientific, #PI-20421) at 4° C. overnight.

The agarose is washed twice with lysis buffer and the Ret proteins areeluted by heating at 98° C. for 5 minutes in 1×NuPAGE LDS sample buffer(Invitrogen, #NP0007). The eluted proteins are separated byelectrophoresis on a Tris-Bis gel (NuPAGE Bis-Tris 4-12% Gel, 1.0 mm, 15well, Invitrogen, #NP0323BOX) and transferred to an Invitrolon PVDFmembrane (pore size 0.45 μm, Invitrogen, #LC2005). The membrane isblocked for 1 hour in 1×TBST containing 5% milk. The membrane is probedovernight at 4° C. with anti-phosphotyrosine(4G10) mouse monoclonalantibody (Millipore Corporation, Cat# 05-321, 1:5,000) in 1×TBST+5%milk.

After washing with 1×TBST for 2 hours with five buffer changes, themembrane is probed with Goat anti-Mouse IgG HRP antibody (JacksonImmunoresearch Labs, #115-035-146, 1:2000) in 1×TBST+5% milk for 1 hourat room temperature. The membrane is washed with TBST for 2 hours withfive buffer changes, treated with ECL plus Western Blot detectionreagent (GE Healthcare, formerly Amersham, #RPN2132) according to theinstruction manual, and the chemiluminescent signal is detected on KodakBiomax MR Film (VWR, # IB8701302).

Biochemical Ret Assay

Compounds are tested for the ability to inhibit Ret kinase in anELISA-based assay. The assay is carried out in a Costar white 96 wellplate (Fisher Scientific, #07-200-591) coated overnight with 0.01 mg/mLNeutrAvidin (Pierce, 100 μL/well) at 4° C. The pre-coated 96 well plateis blocked with 2% BSA in PBST buffer for at least 1 h at roomtemperature before starting the assay. Serially diluted test compoundstock solution is prepared separately in DMSO solution starting from 300μM, and 2 μL/well of this diluted compound (3% DMSO final concentration)is added directly to the NeutrAvidin coated assay plate containing 55.5μL/well of kinase reaction buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 1 mMDTT, 0.01% Brij-35) pre-mixed with ATP and kinase substrate (TK2peptide). Reaction is initiated by adding 2.5 μL/well Ret kinase(Millipore, #14-570) resulting in a final reaction volume of 60 pt. Thereaction is allowed to continue for 30 minutes at room temperature.Final enzyme reaction conditions in 60 μL are 20 mM HEPES, pH 7.4, 5 mMMgCl₂, 1 mM DTT, 0.01% Brij-35, 0.15 ng Ret, 2 μM ATP, 2 μM peptide TK2substrate (Biotin-EGPWLEEEEEAYGWMDF-CONH₂, Anaspec, #60329-1). Aftercompleting the reaction, the wells are washed three times with PBST andincubated for 1 h at room temperature with 100 μL/well of phosphopeptidedetection antibody solution (mixture of 1:10000 diluted mouse anti-pTyrmonoclonal antibody (Cell Signal Technology, #9411) and 1:10000 dilutedgoat HRP-conjugated anti-mouse IgG (Jackson Immunoresearch,#115-035-003)). The plate is washed three times with PBST, developedwith supersignal ELISA pico chemiluminescent substrate (Pierce), andread on a SpectraMax M5 microplate reader (Molecular Devices).

Results

Results for the tryptase, FP-based Syk, CD63, VEGFR and Ret assays arepresented in Table 2 for Compound 1,N4-(2,2-Dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(6-morpholinopyridin-3-yl)-2,4-pyrimidinediamine,Compound 3,N4-(2,2-Dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(4-morpholinophenyl)-2,4-pyrimidinediamine,and a control compound,N4-(2,2-Dimethyl-3-oxo-4H-5-pyrido[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine.

TABLE 2 CD63, FP- FACS, LD based Whole Tryptase Biochem blood CellularBiochem Cellular Biochem Compound CHMC Syk IgE VEGFR2 VEGFR2 Ret Ret

0.04 0.02 0.93 0.48 0.04 0.3 0.002

0.09 0.04 0.25 16.76 0.77 >5 0.048

0.08 0.08 0.30 5.23 0.34 >1 0.006

The LD tryptase, FP-based biochemical, and CD63 assays are on-targetassays. Although Compounds 1 and 3 are somewhat less potent than thecontrol in the whole cell and biochemical assays, they are more potentin the presence of whole blood (the CD63 assay). Without being limitedto any particular theory, Applicants believe that the potency ofCompounds 1 and 3 in the presence of whole blood is due to high specificbinding as compared to high non-specific binding by the control.

The VEGF assay data are significant because, without being limited toany particular theory, Applicants currently believe that VEGF inhibitionleads to elevated blood pressure (see Kamba et al., British Journal ofCancer 96:1788-1795 (2007); Roodhart et al. Current ClinicalPharmacology 3:132-143 (2008); Franklin et al. JPET 329:928-937 (2009)).Compounds 1 and 3 have significantly increased selectivity against VEGFas compared to the control, and therefore have a substantially reducedrisk of hypertension.

Ret kinase is believed to be necessary for kidney development. Becausemore arthritis patients are women than men, any potential developmentaltoxicity, such as may be associated with Ret inhibition, is a seriouslimitation (see Clemens et al. Birth defect Research (Part A) 85:130-136(2009)). Compounds 1 and 3 are significantly more selective over Retkinase than the control.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

We claim:
 1. A method of treating or preventing a disease in a subject,where the disease is selected from an allergic disease, low gradescarring, a disease associated with tissue destruction, a diseaseassociated with tissue inflammation, inflammation and scarring,comprising administering to the subject a pharmaceutically effectiveamount of a compound of any of formulae (I)-(III):

or a pharmaceutically acceptable salt thereof, effective to inhibit theFc receptor signal transduction cascade.
 2. The method according toclaim 1, comprising administering to the subject a pharmaceuticallyeffective amount of the compound of formula (I):

or a pharmaceutically acceptable salt thereof.
 3. The method accordingto claim 1, comprising administering to the subject a pharmaceuticallyeffective amount of the compound of formula (II):

or a pharmaceutically acceptable salt thereof.
 4. The method accordingto claim 1, comprising administering to the subject a pharmaceuticallyeffective amount of the compound of formula (III):

or a pharmaceutically acceptable salt thereof.
 5. A method according toclaim 1, comprising administering to the subject a pharmaceuticallyeffective amount of a pharmaceutically acceptable salt of a compound ofany of formulae (I)-(III).
 6. A method according to claim 1, comprisingadministering to the subject a pharmaceutically effective amount of amono- or di-sodium salt, a mono- or di-potassium salt, a mono- ordi-lithium salt, a calcium salt, a magnesium salt, or an ammonium saltof a compound of any of formulae (I)-(III).
 7. A method according toclaim 1, comprising administering to the subject a pharmaceuticallyeffective amount of a mono- or di-trifluoroacetic acid salt, ap-toluenesulfonic acid salt, a hydrochloride salt, a benzenesulfonicacid salt, or an ethanesulfonic acid salt of a compound of any offormulae (I)-(III)
 8. A method according to claim 1, comprisingadministering to the subject a pharmaceutically effective amount of apharmaceutical composition comprising a compound of any of formulae(I)-(III) or a pharmaceutically acceptable salt thereof, and anacceptable carrier, excipient and/or diluent.
 9. The method according toclaim 1 in which the disease is rheumatoid arthritis.
 10. The methodaccording to claim 9 in which the amount of the compound or saltadministered is effective to achieve a serum concentration of thecorresponding drug that is at or above the IC₅₀ of Syk inhibition of thedrug, as measured in an in vitro assay.
 11. A method of treating orpreventing an autoimmune disease in a subject, and/or one or moresymptoms associated therewith, comprising administering to the subject apharmaceutically effective amount of a compound of any of formulae(I)-(III):

or a pharmaceutically acceptable salt thereof, effective to treat orprevent the autoimmune disease.
 12. The method according to claim 11 inwhich the autoimmune disease is selected from autoimmune diseases thatare frequently designated as single organ or single cell-type autoimmunedisorders and autoimmune disease that are frequently designated asinvolving systemic autoimmune disorder.
 13. The method according toclaim 12 in which the autoimmune disease is selected from Hashimoto'sthyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritisof pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis,Goodpasture's disease, autoimmune thrombocytopenia, sympatheticophthalmia, myasthenia gravis, Graves' disease, primary biliarycirrhosis, chronic aggressive hepatitis, ulcerative colitis, membranousglomerulopathy, systemic lupus erythematosis, rheumatoid arthritis,Sjogren's syndrome, Reiter's syndrome, polymyositis-dermatomyositis,systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullouspemphigoid.
 14. A method of treating a cell proliferative disorder in asubject, comprising administering to a subject suffering from rheumatoidarthritis a pharmaceutically effective amount of a compound of any offormulae (I)-(III):

or a pharmaceutically acceptable salt thereof.
 15. The method accordingto claim 14 in which the cell proliferative disorder is a hematopoieticneoplasm.
 16. The method according to claim 14 in which the cellproliferative disorder is a myeloid neoplasm.
 17. The method accordingto claim 14 in which the proliferative disorder is a virally-mediatedtumor arising from modulation of Syk kinase activity.